PIN1 is recently identified as a peptidyl-prolyl cis/trans isomerase (PPIase). It binds to and isomerizes specific pSer/Thr-Pro motifs and catalytically induces conformational changes after phosphorylation. PIN1 plays an important role in many cellular events, such as cell cycle progression, transcriptional regulation, RNA processing, and cell proliferation and differentiation. There are few reports mentioned about the expression of Pinl in osteosarcoma tissue and the relationship with cyclinD1. So in the current study, we investigated the expression pattern of PIN1 in human osteosarcoma tissues and the role in osteosarcoma generation and development. The expression level of PIN1 was detected by immunohistochemistry and western blotting. Results showed that the expression of PIN1, cyclin D1, and β-catenin were significantly higher in human osteosarcoma tissues compare to normal tissues. The effects of PIN1 overexpression were studied on human osteosarcoma cell lines in vitro. Adenovirus-mediated PIN1 overexpression significantly stimulated the proliferation of MG-63 and U2-OS osteosarcoma cells by 148 ± 10.5% and 187 ± 21.5%, respectively. In FACS analysis, U2-OS cells showed a significant arrest in cell cycle progression at G0/G1. Consistent with increased cell growth, levels of cyclin D1 and cyclin E and their associated cyclin-dependent kinases, CDK4 and CDK6, were enhanced in PIN1 over-expressing cells compared with empty vector-transfected cells. When PIN1 inhibitor juglone was added to the cells, cell proliferating effects of PIN1 were abolished. These results suggest that PIN1 may play an important role in tumorigenesis and tumor progression of osteosarcoma and therefore may provide a new target for gene therapy.