Tripartite motif (TRIM) 31, a member of the TRIM protein family, contributes to a wide range of biological processes and its altered expression exerts a non-negligible effect on multiple pathological conditions such as immunological disorders and retroviral protective activity. Recently, increasing evidence has demonstrated an important role of TRIM31 in the development of various cancers. However, the role of TRIM31 in gallbladder cancer (GBC) remains unclear. In this study, we showed that TRIM31 was elevated in GBC tissues and cell lines and associated with the clinicopathological features of GBC patients. Down-regulation of TRIM31 suppressed GBC cell proliferation, migration and invasion in vitro as well as inhibited tumor growth and metastasis in vivo. In addition, knockdown of TRIM31 reduced the expression of MMP2, MMP9 and p-Akt. Taken together, these findings indicated that knockdown of TRIM31 suppressed proliferation and invasion of GBC cells and was associated with PI3K/Akt signaling inactivation. Thus, TRIM31 may be a potential therapeutic target for the treatment of GBC.