The gene encoding myopodin, an actin binding protein, is commonly deleted in invasive, but not in indolent, prostate cancers. There are conflicting reports on the effects of myopodin expression on prostate cancer cell migration and invasion. The recent recognition that myopodin is expressed as four different isoforms further complicates our understanding of how this potentially important invasive prostate cancer biomarker affects tumor cell migration and invasion. We now show that myopodin affects the chemokinetic, rather than the chemotactic, properties of PC3 prostate cancer cells. Furthermore, all myopodin isoforms can either increase or decrease PC3 cell migration in response to different chemokinetic stimuli. These migration properties were reflected by differences in cell morphology and the relative dependence on Rho-ROCK signaling pathways induced by the environmental stimuli. Truncation analysis determined that a unique 9-residue C-terminal sequence in the shortest isoform and the conserved, PDZ domain-containing N-terminal region of the long isoforms both contribute to the ability of myopodin to alter the response of PC3 cells to chemokinetic stimuli. Matrigel invasion assays also indicated that myopodin primarily affects the migration, rather than the invasion, properties of PC3 cells. The correlation between loss of myopodin expression and invasive prostate cancer therefore reflects complex myopodin interactions with pathways that regulate the cellular migration response to diverse signals that may be present in a tumor microenvironment.