The calcium-binding protein S100A4 has been characterized as a metastasis-inducing molecule, and regulates cell motility and invasiveness of cancer cells. In order to clarify the significance of the expression of S100A4 as a prognostic factor in gallbladder cancer, S100A4 expression in resected gallbladder cancers were examined using an immunohistochemical staining technique. The relationship between S100A4 expression and clinicopathological factors including prognosis were evaluated. Twenty-five of 60 cases (42%) demonstrated positive staining for S100A4. There was no statistically significant association between S100A4 and histological grade, T, N, M factor, presence of stone, or stage. Kaplan-Meier method showed the 5-year survival rate of the group staining positive for S100A4 (31.5%) to be statistically poorer than that of the group staining negative for S100A4 (78.2%). Also in T2 cases, the 5-year survival rate of the group staining positive for S100A4 (57.1%) was statistically poorer than that of the group staining negative for S100A4 (83.3%). On univariate analysis, positive staining for S100A4 was a significant prognostic factor, and the hazard ratio was 4.05. On multivariate analysis, positive staining for S100A4 is also a significant predictor of prognosis second to T factor. These results indicate that positive staining for S100A4 is useful in assessing the prognosis of patients with gallbladder cancer as well as TNM factors.