Tripartite motif-containing protein 65 (TRIM65) is an E3 ubiquitin ligase and a critical regulator of a variety of cellular processes as well as tumor progression. Therefore, more substrates must be identified in the physiology or disease context. Here, we found that TRIM65 is upregulated and associated with poor survival in colorectal cancer (CRC). More specifically, high expression of TRIM65 is associated with CRC metastasis and recurrence. Ectopic overexpression of TRIM65 in CRC cell lines enhanced proliferation, invasion, and migration, while knockdown of TRIM65 expression had the opposite effects. Furthermore, we identified a new substrate of TRIM65, namely ARHGAP35, a Rho GTPase-activating protein (GAP) that is involved in polarized cell migration. Phenotypically, forced expression of TRIM65 induces increased production of migration-related structures, focal adhesions, and/or filopodia and enhances CRC metastasis to the liver or the lung in a mouse model. Mechanistic studies revealed that TRIM65 mediates ubiquitination of ARHGAP35, whose degradation leads to elevated Rho GTPase activity. In addition, we identified several phosphorylation sites on TRIM65. In sum, we reveal a novel TRIM65-GAP-Rho regulatory axis that modulates the actin cytoskeleton and the migration behavior of CRC cells, and the TRIM65-ARHGAP35 interaction might be a valuable therapeutic target in CRC.