The present study demonstrated that invadopodia are associated with invasion by degradation of matrix in prostate cancer cells PC3. To find out the presence of invadopodia in PC3 cells, we performed a few comparative analyses with osteoclasts, which utilize podosomes for migration. Our investigations indeed demonstrated that invadopodia are comparable to podosomes in the localization of Wiskott-Aldrich syndrome protein (WASP)/matrix metalloproteinase-9 and the degradation of matrix. Invadopodia are different from podosomes in the localization of actin/vinculin, distribution during migration, and the mode of degradation of extracellular matrix. Invadopodia enable polarized invasion of PC3 cells into the gelatin matrix in a time-dependent manner. Gelatin degradation was confined within the periphery of the cell. Osteoclasts demonstrated directional migration with extensive degradation of matrix underneath and around the osteoclasts. A pathway of degradation of matrix representing a migratory track was observed due to the rearrangement of podosomes as rosettes or clusters at the leading edge. Reducing the matrix metalloproteinase-9 levels by RNA interference inhibited the degradation of matrix but not the formation of podosomes or invadopodia. Competition experiments with TAT-fused WASP peptides suggest that actin polymerization and formation of invadopodia involve the WASP-Arp2/3 complex pathway. Moreover, PC3 cells overexpressing osteopontin (OPN) displayed an increase in the number of invadopodia and gelatinolytic activity as compared with PC3 cells and PC3 cells expressing mutant OPN in integrin-binding domain and null for OPN. Thus, we conclude that OPN/integrin alphavbeta3 signaling participates in the process of migration and invasion of PC3 cells through regulating processes essential for the formation and function of invadopodia.