Abstract: ObjectiveTo study the effects of p65 microRNA (miRNA) on cell cycle distribution of human breast cancer cell line MDA-MB-231. Methodsp65-targeted miRNA was designed and transfected into MDA -MB-231 cells via lipofecta mineTM2000 mediation. The level of p65 mRNA expression was detected by RT-PCR. NF-κB binding activity of MDA-MB-231 cells was measured by electrophoresis' mobility shift assay (EMSA). Cell cycle distribution was further detected by flow cytometry (FCM).Expression of cyclinD1, CDK4 and p21 proteins were determined by Western blot after transfection. Resultsp65 miRNA expression plasmid was constructed successfully and led to decrease expression level of p65 mRNA dramatically in MDA-MB-231 cells. EMSA assay showed that NF-κB binding activity of MDA-MB-231 cells was inhibited significantly, and lower than that of miR-neg and blank control group (P<0.05). 48 h after transfection, p65 miRNA increased the cell number in G0/G1 phase and decreased the cell proportion in S and G2/M phase (P<0.05). Western blot analized results furtherly confirmed that cyclinD1 protein was inhibited in p65 miRNA-transfected cells, while p21 protein increased after transfection. ConclusionSilencing p65 gene by miRNA was able to arrest MDA-MB-231 cells phase at G0/G1 phase, which might be mediated by downregulation of cyclinD1 and upregulation of p21.