Prolactin (PRL) is recognized as a growth and differentiating hormone in the human breast. These effects are mediated by the PRL receptor (PRLr); when stimulated the PRL-PRLr complex activates several signaling cascades, including those involving the GTP-binding proteins Ras and Rac. The activation of these signaling pathways has been associated with cytoskeletal alterations and increased cellular motility. We hypothesized that such changes could occur in PRL-stimulated human breast cancer cells. To test this hypothesis, complementary studies, including wound closure, time-lapse video microscopy (TLVM), and Boyden chamber assay were performed. These studies revealed that PRL significantly enhanced the migration of the breast cancer cell lines T47D, MCF7, and MDA23 1. Co-stimulation with PRL was noted to potentiate epidermal growth factor (EGF)-induced cell motility. IF microscopy of filamentous actin using rhodamine-conjugated phalloidin revealed a significant and rapid generation of both membrane ruffling and stress fibers in response to PRL, an effect inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In sum, these data reveal that PRL stimulation modulates the cytoskeleton and induces the motility of human breast cancer cells in vitro, events that have been associated with the progression of mammary carcinoma in vivo. Given the recently delineated autocrine-paracrine role for PRL in human breast cancer, these findings could be of appreciable clinical significance.