HPV infection is the main risk factor for cervical cancer, but it is still an insufficient event for the development of this cancer. It has demonstrated that nicotine plays an important role in cervical carcinogenesis. However, the effects and mechanisms of nicotine stimulation on precancerous lesions of cervical cancer are not clear. In this study, it showed that nicotine significantly promoted H8 cells proliferation in a concentration-dependent manner. Moreover, nicotine decreased the level of P53, resulted from a shortened P53 half-life. Note as well that level of P21 protein were decreased along with P53 in a concentration dependent change. It suggested that reduction in stabilization of P53 induced by nicotine may be negative regulator for P53/P21 signaling pathway that acts to prevent the growth of cells. In addition, reduction of RPS27a expression in nicotine treatment H8 cells up-regulated phosphorylation of Mdm2 at serine residue 166, followed by facilitating Mdm2-mediated ubiquitination of P53. Simply put, these findings suggest that nicotine promotes H8 cell lines proliferation by activating RPS27a-Mdm2-P53 pathway in vitro.