Colorectal cancer (CRC) is one of the most common malignancies in the world. Emerging evidence has shown that dysregulation of tripartite motif (TRIM) family proteins is strongly correlated with the tumorigenesis of CRC. Here, we evaluated the biological roles of TRIM66, a member of TRIM family, in the progression of CRC. The results demonstrated that TRIM66 was markedly up-regulated in both CRC tissues and cell lines. To further investigate the functions of TRIM66 in CRC, CRC cells were infected with lentivirus expressing anti-TRIM66 shRNA (sh-TRIM66) or control lentivirus (sh-con). We found that knockdown of TRIM66 significantly inhibited cell proliferation, migration, invasion of CRC cells. TRIM66 knockdown also suppressed epithelial-mesenchymal transition (EMT), as proved by the increased E-cadherin expression and decreased expressions of N-cadherin and vimentin. Furthermore, TRIM66 knockdown markedly inhibited tumor growth in a mouse xenograft model. Knockdown of TRIM66 reduced the activation of JAK2/STAT3 signaling pathway in CRC cells. Treatment with AG490, an inhibitor of JAK2/STAT3 signaling pathway, enhanced the inhibitory effects of TRIM66 knockdown on cell proliferation, migration and invasion. These findings suggested that knockdown of TRIM66 exhibited anti-tumor activity through inhibiting the JAK2/STAT3 signaling pathway in CRC cells.