Induction of the apoptosis of tumor cells is a promising therapeutic approach for the treatment of cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a novel type of anticancer drug. However, gallbladder cancer cells (GBC) exhibit strong resistance to TRAIL. The aim of the present study was to assess the effect of rocaglate CR-1-31B (CR-31), an inhibitor of eukaryotic translation initiation factor 4A (eIF4A), on the sensitization of cells to TRAIL-induced apoptosis in TRAIL-resistant GBC. eIF4A was highly abundant in GBC tissues and cell lines (GBC-SD and SGC-996). GBC cells were treated using TRAIL and/or CR-31 and then apoptosis and TRAIL signaling were detected in vitro. CR-31 enhanced the sensitivity of TRAIL-resistant GBC cells, due to the CR-31-mediated eIF4A translational downregulation of c-FLIP and the subsequent activation of the caspase cascade. Furthermore, GBC-SD tumor xenografts models were established and the effects of CR-31 in vivo were assessed. CR-31 significantly reduced the growth and initiated the apoptosis of tumor cells, suggesting that CR-31 also increased sensitivity in vivo. Taken together, the results of the present study show that CR-31 treatment countered the resistance to TRAIL in GBC cells in vitro and in vivo. Therefore, eIF4A may serve as a novel therapeutic target and its combination with TRAIL-CR-31 as a therapy may serve as a novel strategy for GBC treatment.