Caesar made his second invasion of Britain in 54 BC, and achieved some measure of success, advancing as far as modern day Greater London, and restoring a friendly king, Mandubracius of the Trinovantes, whose father had been deposed by his rival Cassivellaunus. Cassivellaunus led the British opposition to Caesar's incursion, but this led to divided loyalties among the Britons, as Caesar records. Emissaries of five British tribes, including the Ancalites (the others being the Cenimagni, the Segontiaci, the Bibroci and the Cassi), arrived at the Roman camp to treat for peace, and revealed to him the location of Cassivellaunus' stronghold. Caesar besieged him there and brought him to terms. When Caesar left Britain he took hostages of the Britons, although whether the Ancalites were compelled to give any is not specified.
Where exactly the Ancalites were based is not known. In the 16th century William Camden reported that "some doe thinke" the Ancalites inhabited the area around Henley, Oxfordshire, and the Wiltshire tourist board claim them amongst Wiltshire's own ethnic ancestry, which would significantly increase their territory, but without archaeological evidence, none of this can be confirmed.