Gallbladder cancer (GBC) is a highly malignant cancer with poor prognosis. Extensive studies have reported the vital functionality of several microRNAs (miRNAs) in numerous human cancers, including GBC. Microarray analysis has identified the differentially expressed miR-551b-3p in GBC. Therefore, this study aims to validate the underlying mechanism by which miR-551b-3p participated in epithelial-mesenchymal transition (EMT), invasion and migration of GBCs. Bioinformatic analysis predicted the binding of miR-551b-3p to H6PD. We validated the reduced miR-551b-3p expression and increased H6PD expression in the GBC tissues and GBC cell lines. Artificial modulation of miR-551b-3p and H6PD (down- and upregulation) was conducted to explore their roles in EMT, invasive, and migratory abilities of GBCs, and the tumor-bearing mice were used to determine tumor growth. Overexpression of miR-551b-3p or silencing of H6PD was observed to suppress the expressions of N-cadherin and vimentin, and to promote the expression of E-cadherin, along with reduced invasive and migratory ability of GBCs. Mechanistically, miR-551b-3p could evidently target and inhibit the expression of H6PD. Moreover, in vivo experiments substantiated the tumor-inhibiting activities of miR-551b-3p in nude mice. Conjointly, our study suggests that overexpression of miR-551b-3p inhibited the EMT, migration, and invasion of GBCs by inhibiting the expression of H6PD, indicating that miR-551b-3p may serve as a potential target for future development of therapeutic strategies for GBC.