Alpha-methyacyl-CoA racemase (AMACR) is a critical peroxisomal and mitochondrial enzyme, encodes a key enzyme in the catabolism of long-chain fatty acid thus is indispensable in the £]-oxidation of fatty acid to generate biological energy. AMACR stands in many organs with only very low expression level and its overexpression is exclusively in neoplastic conditions. Recently, AMACR overexpresison has been discerned to relevant to tumor progression of prostate, gastric, and colon cancers, and its overexpression has now been introduced in pathological differential diagnosis of prostate carcinoma from non-malignant mimickers. By using 89 gallbladder carcinoma (GBCA) samples for AMACR immunostaining we found AMACR overexpression is frequently discerned in GBCA. It not only significantly correlates with numerous adverse clinicopathologic factors but also manifests a significant independent predictor of worse outcome in GBCA patients. In multivariate comparison, higher tumor stage represented the strongest prognosticator (p = 0.0101), followed by old patient age (p = 0.0378). Moreover, AMACR overexpression also identified patients at around 2-fold higher risk of disease-specific death (p = 0.0452). By Western blot analyses, we found AMACR expression in the metastatic cells, RCB1129, was apparently more abundant than that in its primary lesion RCB1130. By XTT analyses, the viability of both RCB1129 and RCB1130 cells were significantly decreased by AMACR inhibitor. The RCB1129 cell line, with more abundant AMACR protein expression, was more resistant to AMACR inhibitor treatment than RCB1130 cell line at various drug concentrations. Our data suggest AMACR is a prognostic marker that can serve as a promising therapeutic target in gallbladder cancer.