The interaction between tumor cells and extracellular matrix (ECM) proteins influences cell migration and the invasive behavior of cancer cells. In this study, we provide experimental evidence that collagen I and fibronectin affect ovarian cancer cell migration. In vitro wound healing assays and transwell migration assays were used to measure both total wound healing and directionality of individually migrating OV2008 and C13 ovarian cancer cells on glass, collagen I and fibronectin. Involvement of p21-activated kinase 2 (Pak2) in the motility of these cell lines was investigated using a chemical inhibitor as well as siRNA transfection. Culturing ovarian cancer cells on collagen type I (COLL) increased the migratory ability of OV2008 and C13 cells by increasing the directional migration of cells. In contrast, fibronectin (FN) decreased the migratory ability of OV2008 cells by reducing their ability to migrate directionally. When both cell lines are cultured on COLL and treated with increasing concentrations of a PAK inhibitor (IPA-3), there is a dose-dependent response such that there is a decrease in migration with an increase in inhibitor concentration. Further experiments utilizing PAK2 knockdown via siRNA transfection demonstrated significantly reduced migration of OV2008 cells on COLL as compared to those receiving control siRNA. In conclusion, our results indicate that FN and COLL affect the motility of the selected ovarian cancer cells lines and the effect of COLL is likely mediated, at least in part, by PAK2. A better understanding of the molecular events that contribute to tumor invasion and metastasis is crucial for developing novel treatment strategies to improve the long-term survival of women with ovarian cancer. As PAK2 is involved in motility, it should be further explored as a pro-metastatic gene in ovarian cancer.