Abstract: Objective To investigate the protein expression of the p16 gene and the methylation of its promoter in breast cancer, and to analyze the correlation between the p16 DNA methylation and the clinicopathological features. Methods Immuno-histochemistry technique (SP method) and methylation-specific-PCR (MSP) were used to detect p16 protein expression and the methylation of the p16 promoter in 47 breast cancer samples as well as in 20 hyperplasia samples of mammary glands. Results The p16 protein expression in breast cancer samples significantly lower when compared with those of hyperplasia samples (48.9% vs. 70.0%) and p16 methylation was more frequent in breast-tumor tissues when compared with those of hyperplasia samples (38.3% vs. 20.0%), but the statistical significance wasn’t found (P>0.05). Down-regulation of p16 protein was negatively correlation with p16 gene hypermethylation (r=-0.33, P=0.02). Meanwhile, p16 methylation in breast cancer tissues correlated with histological type, lymph node metastasis, but not correlated with the age, tumor diameter, TNM stage, expression of estrogen receptor (ER) and progesterone receptor (PR) gene status. Conclusion The downregulation of p16 protein induced by promoter methylation of p16 gene may not contribute to early cancinogenesis, but may contribute to progression of breast cancer.