In this study, a label-free quantitative proteomic approach was employed to analyze the serial passaged human skin fibroblast （CCD-1079Sk） cells, within 3305 proteins quantified. Of which, 372 proteins were significantly changed in early passage(P6), middle passage (P12) and later passage (P21), with a time-dependent decrease or increase tendency. Then, of which, SMC4 was selected for further biological validation. The results confirmed that the expression of SMC4 was significantly down-regulated in a time-dependent manner in the subculture of human skin fibroblasts (HSFb) with Western Blot experiment.