p27 is a member of the Kip family of cyclin-dependent kinase inhibitors and its overexpression results in cell cycle arrest at G1 and/or apoptosis. In addition to its role as a regulator of cell cycle progression, p27 can also participate in cell motility, especially when it is mislocalized in the cytosol. To further elucidate the role of p27 in the motility of MDA-MB-231 breast cancer cells, we performed p27 knockdown in MDA-MB-231 cells by RNA interference. Infection of MDA-MB-231 cells with retroviruses harboring p27 short hairpin RNA (shRNA) designed from human p27 cDNA resulted in efficient inhibition of p27 expression, while p27 shRNA designed from mouse p27 cDNA did not affect p27 expression in MDA-MB-231 cells. MDA-MB-231 cells infected with human p27 shRNA (MDA-MB-231 hp27shRNA) showed increased proliferation compared to control MDA-MB-231 cells and MDA-MB-231 cells infected with mouse p27shRNA (MDA-MB-231 mp27shRNA). Wound healing assays revealed that migration of MDA-MB-231 hpshRNA cells was markedly impaired compared to MDA-MB-231 mpshRNA cells, especially when cycloheximide was added to block protein synthesis. Immunostaining of p27 in MDA-MB-231 cells showed that p27 predominantly localized in the nuclei. These results suggest that both nuclear and cytosolic p27 can promote cancer cell motility.