Previously, we showed that progesterone (P4) inhibits proliferation and migration of rat aortic smooth muscle cells (RASMCs). The P4-induced migration inhibition in RASMCs resulted from suppression of the Ras homolog gene family, member A (RhoA) activity mediated by cSrc activation. We also observed that P4 increased the formation of p27-RhoA complex in RASMCs. The aim of this study was to further study the involvement of p27 in P4-induced migration inhibition in RASMCs. Treatment with P4 (50 nM) increased the level of p27 protein in RASMCs. Knockdown of p27 abolished the P4-induced increases of the levels of p27 protein and decreases of cell migration in RASMCs. We conducted Western blot analyses and applied pharmacologic inhibitors to delineate the signaling pathway involved in the P4-induced p27 up-regulation and migration inhibition in RASMCs. Our data suggest that P4 increased the levels of p27 in RASMCs through activating the cSrc/AKT/ERK 2/p38 pathway mediated by nongenomic progesterone receptor. The findings of the present study highlight the molecular mechanisms underlying P4-induced migration inhibition in RASMCs.