Phospholipase C-gamma1 (PLC-gamma1), which generates two second messengers, namely, inositol-1, 4, 5-trisphosphate and diacylglycerol, is implicated in growth factor-mediated chemotaxis. However, the exact role of PLC-gamma1 in integrin-mediated cell adhesion and migration remains poorly understood. In this study, we demonstrate that PLC-gamma1 is required for actin cytoskeletal organization and cell motility through the regulation of Pyk2 and paxillin activation. After fibronectin stimulation, PLC-gamma1 directly interacted with the cytoplasmic tail of integrin beta1. In PLC-gamma1-silenced cells, integrin-induced Pyk2 and paxillin phosphorylation were significantly reduced and PLC-gamma1 potentiated the integrin-induced Pyk2/paxillin activation in its enzymatic activity-dependent manner. In addition, specific knock-down of PLC-gamma1 resulted in a failure to form focal adhesions dependent on fibronectin stimulation, which appeared to be caused by the suppression of Pyk2 and paxillin phosphorylation. Interestingly, PLC-gamma1 potentiated the activations of Rac, thus integrin-induced lamellipodia formation was up-regulated. Consequently, the strength of cell-substratum interaction and cell motility were profoundly up-regulated by PLC-gamma1. Taken together, these results suggest that PLC-gamma1 is a key player in integrin-mediated cell spreading and motility achieved by the activation of Pyk2/paxillin/Rac signaling.