The prolyl isomerase Pin1, which specifically catalyzes conformational changes in certain proline-directed phosphorylation sites, is thought to be a critical catalyst for multiple oncogenic pathways. However, little is known about the role of Pin1 in human cervical cancer. Our previous study showed that Pin1 was overexpressed in cervical cancer tissues as well as cell lines. In this study, whether Pin1 is involved in cervical oncogenesis by regulating cyclin D1 was explored and the potential of Pin1-targeted gene silencing in inhibiting cellular growth and tumorigenicity in cervical cancer was investigated. A Pin1-directed shRNA and a sense Pin1 plasmid were constructed, and then the effects of the shRNA and the sense plasmid on HeLa cells were evaluated. The results showed that Pin1 directly regulated cyclin D1 levels. In addition, silencing Pin1 with RNAi significantly reduced cancer cell proliferation, colony formation, and strongly enhanced the apoptosis of HeLa cells. It is suggested that Pin1 may contribute to cervical tumorigenesis by regulating cyclin D1 expression and Pin1 may serve as a promising molecular target for diagnostics and therapeutics in cervical cancer.