Cell cycle regulatory proteins are important indicators in determining progression through the cell cycle. Recent experimental evidence shows that active Cdk4-Cyclin D1 complexes help cells to pass through the R point, a point of no return, after which the cells become committed to a new round of replication. It is widely known that P16INK4a can arrest cells in the G1 phase, but how the expression of exogenous P16INK4 gene can affect the activity of Cdk4-Cyclin D1 remains unclear. In this study, using exogenous wild-type P16 gene, antibodies for P16, Cdk4, Cyclin D1 and Rb proteins, and primers for these genes, we examined the expression of exogenous wild-type P16 gene and the changes of cell cycle regulatory genes (Cdk4, Cyclin D1, and Rb) in human bladder cancer cells. The cell cycle analysis revealed that the proliferation of P16 gene-transfected cancer cells was inhibited after the transfection of exogenous wild P16 gene. The immunocytochemical results indicated that after the transfection of exogenous wild-type P16 gene, the expression of Cdk4, Cyclin D1, and Rb were negative in the nuclei, whereas the expression of P16 significantly increased in the nuclei and the cytoplasm. The RT-PCR results showed that the transcription of P16 gene increased significantly after the transfection, whereas the transcription of Cdk4, Cyclin D1, and Rb decreased. Our results suggest that the transfection of exogenous wild P16 gene induces the bladder cancer cells arrested in G0/G1 phase, and the increasing expression of P16 inhibits the expression of Cdk4, Cyclin D1, and Rb in nuclei. As a consequence, exogenous P16 has negative effects on the malignant proliferation of the bladder cancer cells, and it may be considered as target for potential anticancer drugs.