PA 401 begins at an intersection with PA 23 in Caernarvon Township, Berks County, heading southeast on two-lane undivided Conestoga Road. The route heads into agricultural areas as it crosses into Elverson, Chester County, where it runs between farmland to the north and rural areas of woods and homes to the south, crossing PA 82. After this junction, the road runs between housing developments to the north and rural areas to the south, crossing into West Nantmeal Township. PA 401 continues into forested areas with some farm fields as it enters East Nantmeal Township and comes to the PA 345 intersection. At this point, the route continues southeast through more woods before heading into farmland and turning south. The road crosses into forests and turns sharply to the east, entering areas of farms with a few residences. Farther east, PA 401 heads into more wooded areas of homes as it crosses into West Vincent Township and reaches the PA 100 junction.
The route continues southeast past a mix of farms, woods, and housing developments, briefly crossing into Upper Uwchlan Township before heading back into West Vincent Township. The road curves south before continuing southeast into West Pikeland Township and intersecting PA 113. Pa st this intersection, PA 401 passes through wooded areas of residential development, passing under the Pennsylvania Turnpike (I-76). A short distance later, the route comes into Charlestown Township and passes more forested areas of homes before turning east into East Whiteland Township. The route passes more housing developments as it widens to four lanes at the Phoenixville Pike intersection and reaches the interchange with the US 202 freeway, where it is briefly a divided highway. The route narrows back to a two-lane undivided road past this interchange and crosses the Chester Valley Trail before continuing southeast to the eastern terminus at US 30 in commercial areas.
Pennsylvania Route 401 Alternate is a route that circumvents a narrow bridge in Chester Springs, Pennsylvania. It was signed on November 1, 2013 and is partially routed on Byers Road, Graphite Mine Road, PA 100, and PA 113.