Kiefer was taken notice of as an outstanding junior. He won the Junior Australian Open, US Open and was a finalist and semi-finalist at Wimbledon and the French Open finishing as the number 2 junior behind Mariano Zabaleta when he was 18.
On 10 January 2000, he reached his second quarter-final at the Australian Open and afterwards was ranked World No. 4, his highest position to date.
Nicolas has been known to have a few tennis superstitions. He is sometimes seen tapping his racquet on the corners of the court after a point, although the reasons behind this are not clear. He also, when serving, frequently asks for the ball with which he has just won a point to re-use it in the next one.
Kiefer became infamous for an incident on 25 January 2006, during the quarter-finals of the Australian Open. While facing Sébastien Grosjean late in the fifth set of a marathon match, Kiefer threw his racquet midpoint. Grosjean lost the point, hitting the ball into the net. Grosjean protested that the racquet distracted his shot. The umpire Carlos Bernardes said he did not believe the act was intentional and noted Grosjean had already hit the ball before the flying racquet could have had any effect on his shot. Grosjean eventually lost the fifth and final set to Kiefer. Kiefer went through to the semi-finals where he was defeated by the 2004 champion Roger Federer.
Kiefer injured his wrist while playing at the 2006 French Open, and announced his return on 5 July 2007, having fallen to the 404th position on ATP. He announced that he was "tired of waiting and anxious to start traveling again and to see his name on scoreboards". Kiefer returned at the 2007 Gerry Weber Open, losing in the first round to eventual champion Tomáš Berdych. At Wimbledon, he made the third round after defeating No.30 seed Filippo Volandri and Fabrice Santoro, both in straight sets, before losing in 4 sets (3 of which were tiebreakers) to Novak Djoković. At Newport, however, he ended up losing in round 1. At Los Angeles, he reached the semifinals in only his 4th tournament since coming back from injury; he had to default against Radek Štěpánek, another player coming back from injury, because of an injury sustained during his quarter-final win. He also made an impressive showing at the 2007 Madrid Masters, where he beat number five seed Fernando González in the quarterfinals before losing in the semifinals to world number one Roger Federer 6–4, 6–4.
In the 2009 Sony Ericsson Open in Miami, Kiefer beat "the magician" Fabrice Santoro 5–7 7–6(5) 6–2 in Second Round. In Third Round Kiefer was defeated by World No.2 Roger Federer 6–4 6–1. At the 2009 Monte-Carlo Rolex Masters Kiefer lost in his first match against qualifier Andreas Beck 1–6 7–6(1) 6–4. At the 2009 Internazionali BNL d'Italia in Rome Kiefer lost again in the first match against Juan Mónaco in straight sets 6–2 6–3. In the 2009 BMW Open Kiefer was down against Ernests Gulbis 2–6 0–2 but eventually won 2–6 6–3 6–2. Kiefer said after the match, "Clay and me, we will never be the best of friends". Kiefer suffered from problems in his back which eventually made him lose against Jérémy Chardy in the next round 6–4 7–6(9). At the 2009 Mutua Madrileña Madrid Open he lost against Tommy Robredo 3–6 6–2 6–2. Kiefer then played the 2009 ARAG World Team Cup in which he played the doubles matches together with Mischa Zverev. They won all of their matches and Germany reached the final but lost against Serbia as Germany lost both of their single matches. Despite Germany losing, Kiefer won the doubles match in the final against Viktor Troicki and doubles World No.1 Nenad Zimonjić 7–5 4–6 10–7. Kiefer then participated at the 2009 French Open in which he beat qualifier Ilija Bozoljac in 4 sets 7–6(4) 3–6 7–5 6–4. However, Kiefer lost in the Second Round against World No.14 David Ferrer in 5 sets 6–3 5–7 6–4 3–6 6–2. Despite this loss, Kiefer claimed that he was proud that he had played up to a fifth set against one of the best tennis players of the world in clay, since clay is Kiefer's least favourite surface. The clay season had now ended, and the grass season started with Kiefer's participation in his favourite tournament, the 2009 Gerry Weber Open. In the first match he trashed Viktor Troicki 6–2 6–1, but retired in the Second Round against Jürgen Melzer when he was down 6–1 since he had a muscular strain in his abdomen which forced him to retire not only from the match but from the doubles where he had reached the semifinals with Mischa Zverev. Kiefer participated at the 2009 Wimbledon Championships despite having not fully recovered from his abdomen injury. This was reflected in his match against Fabrice Santoro where Kiefer lost in straight sets 6–4 6–2 6–2. Kiefer then played for Germany in the 2009 Davis Cup quarter-finals against Spain. He did so in the doubles match together with Mischa Zverev against Spain's doubles players Fernando Verdasco and Feliciano López. Kiefer and Zverev lost the match 6–3, 7–6(1), 6–7(6), 6–3. In the 1st round of the U.S Open he beat Michaël Llodra 6–3, 6–4, 6–4, but in the second round he lost to number 3 ranked Rafael Nadal 0–6, 6–3, 3–6, 4–6.