Proline-rich protein tyrosine kinase 2 (Pyk2) is a member of focal adhesion kinase family. We studied Pyk2 role in cutaneous wound repair using Pyk2-null mice. We report that the rate of wound closure was delayed in Pyk2-null compared with control mice. To examine whether impaired wound healing of Pyk2-null mice was caused by a keratinocyte cell-autonomous defect, the capacities of primary keratinocytes from Pyk2-null and wild-type (WT) littermates to heal scratch wounds in vitro were compared. The rate of scratch wound repair by Pyk2-null keratinocytes was decreased compared with that by WT cells. Moreover, cultured human epidermal keratinocytes overexpressing dominant-negative mutant of Pyk2 failed to heal scratch wounds. Conversely, stimulation of Pyk2-dependent signaling via WT Pyk2 overexpression induced accelerated scratch wound closure, and was associated with increased expression of matrix metalloproteinases (MMPs) -1, -9, and -10. The Pyk2-stimulated increase in the rate of scratch wound repair was abolished by co-expression of dominant-negative mutant of Protein Kinase C (PKC) and by GM-6001, a broad spectrum inhibitor of MMP activity. These results suggest that Pyk2 is essential for skin wound re-epithelialization both in vivo and in vitro, and that it regulates epidermal keratinocyte migration via a pathway that requires PKC and MMP functions.