Previously, we reported that fenvalerate (Fen) promotes proliferation of human uterine leiomyoma (UtLM) cells by enhancing progression of cells from G(0)-G(1) to S phase through molecular mechanisms independent of estrogen receptor-α and -β. The cyclin-dependent kinase (CDK) inhibitor p27, which blocks G(1) to S phase transitions and is an important regulator of CDK2, is often decreased in hormonally regulated diseases, including uterine leiomyomas. Therefore, we were interested in whether Fen could regulate the expression of p27 and whether p27 might play a role in Fen-induced cell proliferation. Expression of p27 in Fen-treated UtLM and uterine smooth muscle cells (UtSMCs) was examined. We found that p27 mRNA was significantly downregulated and that protein levels were decreased in both cell types treated with 10 μM Fen for 24 h compared with respective controls. Overexpression of p27 in UtLM cells and UtSMCs using an adenovirus doxycycline (Dox)-regulated Tet-off system abrogated the proliferative effects of Fen, as evidenced by decreased total cell numbers and BrdU incorporation. Fen treatment increased CDK2 mRNA expression levels; however, overexpression of p27 also abolished this effect. In contrast, Dox treatment dramatically restored the above muted responses. Finally, we utilized siRNA to knock down p27 expression. After transfection, mRNA levels of p27 were downregulated in UtLM cells and UtSMCs and total cell numbers and BrdU incorporation increased significantly compared with nontransfected cells. Fen treatment in the presence of p27 silencing enhanced the increased cell counts and BrdU labeling in UtLM cells and UtSMCs. Taken together, these results indicate that p27 downregulation is critical for Fen-induced cell proliferation.