Gallbladder cancers have a very poor prognosis without specific molecular marker being identified. In this study we studied PUMA, c-Myb and p53 expression in benign and malignant lesions of gallbladder and analyzed their clinicopathological significance.Immunohistochemical staining of PUMA, c-Myb and p53 protein was performed in 108 gallbladder adenocarcinomas, 46 peritumoral tissues, 15 polyps, and 35 chronic cholecystitis.We demonstrated that the percent of positive PUMA, c-Myb and p53 expression was significantly higher in gallbladder adenocarcinomas than in peritumoral tissues, polyps and chronic cholecystitis (p < 0.05 or 0.01). Benign gallbladder epithelium with positive PUMA, c-Myb or p53 expression showed moderately or severely atypical hyperplasia. The percent of positive PUMA, c-Myb and p53 expression was significantly higher in the cases having poorly differentiated adenocarcinoma with large tumor mass, lymph node metastasis and high invasiveness than cases with well-differentiated adenocarcinoma with small tumor mass and without metastasis and invasiveness (p < 0.05 or p < 0.01). The percent of positive PUMA, c-Myb and p53 expression was significantly higher in cases with radical resection than without resection (p < 0.05). Univariate Kaplan-Meier analysis showed that PUMA, c-Myb and p53 expression was associated with decreased overall survival (p < 0.05 or p < 0.01). Multivariate Cox regression analysis showed that PUMA, c-Myb or p53 expression was a poor-prognostic predictor in gallbladder adenocarcinoma.PUMA, c-Myb and p53 expression closely relates to the carcinogenesis, fast-progression, easy-metastasis, high-invasion, and poor-prognosis in gallbladder adenocarcinoma.