Colorectal cancer (CRC) is one of the most prevalent cancers worldwide and is typically treated with the FOLFOX regimen (folinic acid, 5-fluorouracil, and oxaliplatin). However, oxaliplatin resistance remains a serious clinical problem. In the present study, we found that SUMO2/3 was overexpressed in CRC tissues and exogenous overexpression of SUMO2/3 promoted CRC cell proliferation, extension, and invasion and positively regulated the cell cycle. In contrast, SUMO2/3 gene knockdowns inhibited migration and repressed cell viability in vitro and in vivo. In addition, we found that SUMO2/3 was recruited to the cell nucleus and suppressed oxaliplatin-induced apoptosis of CRC cells. Moreover, Ku80, a DNA-binding protein essential for the repair of DNA double-strand breaks, was confirmed to bind with SUMO2/3. Notably, Ku80 undergoes SUMOylation at K307 by SUMO2/3 and this correlated with apoptosis in CRC cells suffering oxaliplatin stress. Collectively, we found that SUMO2/3 plays a specific role in CRC tumorigenesis and acts through Ku80 SUMOylation which is linked with the development of CRC-oxaliplatin resistance.© 2023 International Union of Biochemistry and Molecular Biology.