Cell adhesions play an important role in neurite extension. Paxillin, a focal adhesion adaptor protein involved in focal adhesion dynamics, has been demonstrated to be required for neurite outgrowth. However, the molecular mechanism by which paxillin regulates neurite outgrowth is unknown. Here, we show that paxillin is phosphorylated by p38MAPK in vitro and in nerve growth factor (NGF)–induced PC-12 cells. Ser 85 (Ser 83 for endogenous paxillin) is identified as one of major phosphorylation sites by phosphopeptide mapping and mass spectrometry. Moreover, expression of the Ser 85 → Ala mutant of paxillin (pax S85A ) significantly inhibits NGF-induced neurite extension of PC-12 cells, whereas expression of wild-type (wt) paxillin does not influence neurite outgrowth. Further experiments indicate that cells expressing pax S85A exhibit small, clustered focal adhesions which are not normally seen in cells expressing wt paxillin. Although wt paxillin and pax S85A have the same ability to bind vinculin and focal adhesion kinase, wt paxillin more efficiently associates with Pyk2 than pax S85A. Thus, phosphorylation of paxillin is involved in NGF-induced neurite extension of PC-12 cells, probably through regulating focal adhesion organization.