This study was conducted to evaluate the prognostic role of fascin expression in gallbladder (GB) cancer and to define the relationship of thrombospondin-1 (TSP-1) and syndecan-1 in fascin expression. We performed immunohistochemical detection of fascin, TSP-1, and syndecan-1 in 43 tissue samples from GB cancer patients who underwent macroscopic complete resection. There were 19 (44%) and 24 (56%) cases having low- and high-grade fascin expression, respectively. The tumors with high-grade fascin expression tended to more frequently show poorer differentiation, deeper invasion depth, lymph node metastasis, a higher American Joint Committee on Cancer stage, and recurrence (each P < 0.05). The patients with high-grade fascin expression had significantly shorter survival periods than those with low-grade fascin expression (P < 0.05). The frequency of positive TSP-1 or syndecan-1 expression in the cases with high-grade fascin expression was significantly higher than that in the cases with low-grade fascin expression (each P < 0.05). These results suggest that a subset of advanced GB cancers revealed a marked overexpression of fascin, which was associated with aggressive clinicopathologic findings and poor overall survival. Furthermore, fascin, TSP-1, and syndecan-1 may act in concert to mediate a more aggressive clinical course through enhanced tumor cell motility.