Gallbladder cancer, the most common biliary tract malignancy, is a highly malignant neoplasm. In the present work, we have analyzed the significance of cell cycle-related proteins to predict prognosis and to provide guidance for optimal therapeutic decision-making in patients with gallbladder adenocarcinoma. The expressions of p16, p21, p27, p53, p63, cyclin D1, bcl-2 and bcl-6 were examined in a tissue microarray constructed from 96 cases of gallbladder adenocarcinoma by immunohistochemistry and correlated with clinicopathologic prognostic factors. Expression of p16 was correlated with a low pT stage, adenoma background and good prognosis. Cases with p63 expression showed a higher T stage, more frequent perineural invasion and poor prognosis when compared to cases without p63 expression. Over-expression of p53 or loss of p53 was associated with poor tumor differentiation, frequent distant metastasis and low disease-specific survival rate. The expressions of p21, p27, bcl-2, bcl-6 and cyclin D1 were not significant prognostic factors for gallbladder adenocarcinoma. These results indicate that p16, p63 and p53 can be used as prognostic markers in gallbladder adenocarcinoma; especially p53 and p63 as poor prognostic markers and p16 as a favorable prognostic marker.