AbstractSumoylation is an essential posttranslational modification in eukaryotes that has emerged as an important pathway in oncogenic processes. Most human cancers display hyperactivated sumoylation and many cancer cells are remarkably sensitive to its inhibition, thus supporting application of chemical sumoylation inhibitors in cancer treatment. Here we show, first, that transformed embryonic fibroblasts derived from mice haploinsufficient for Ubc9, the essential and unique gene encoding the SUMO E2 conjugating enzyme, exhibit enhanced proliferation and transformed phenotypes in vitro and as xenografts ex vivo. To then evaluate the possible impact of loss of one Ubc9 allele in vivo, we used a mouse model of intestinal tumorigenesis. We crossed Ubc9^+/− mice with mice harboring a conditional ablation of Apc either all along the crypt–villus axis or only in Lgr5⁺ crypt-based columnar (CBC) cells, the cell compartment that includes the intestinal stem cells proposed as cells-of-origin of intestinal cancer. While Ubc9^+/− mice display no overt phenotypes and no globally visible hyposumoylation in cells of the small intestine, we found, strikingly, that, upon loss of Apc in both models, Ubc9^+/− mice develop more (>2-fold) intestinal adenomas and show significantly shortened survival. This is accompanied by reduced global sumoylation levels in the polyps, indicating that Ubc9 levels become critical upon oncogenic stress. Moreover, we found that, in normal conditions, Ubc9^+/− mice show a moderate but robust (15%) increase in the number of Lgr5⁺ CBC cells when compared to their wild-type littermates, and further, that these cells display higher degree of stemness and cancer-related and inflammatory gene expression signatures that, altogether, may contribute to enhanced intestinal tumorigenesis. The phenotypes of Ubc9 haploinsufficiency discovered here indicate an unanticipated tumor-suppressive role of sumoylation, one that may have important implications for optimal use of sumoylation inhibitors in the clinic.