Colorectal cancer is the third most common cancer worldwide. Although the transcription factor c-MYC is misregulated in the majority of colorectal tumors, it is difficult to target directly. The deubiquitinase USP28 stabilizes oncogenic factors, including c-MYC; however, the contribution of USP28 in tumorigenesis, particularly in the intestine, is unknown. Here, using murine genetic models, we determined that USP28 antagonizes the ubiquitin-dependent degradation of c-MYC, a known USP28 substrate, as well as 2 additional oncogenic factors, c-JUN and NOTCH1, in the intestine. Mice lacking Usp28 had no apparent adverse phenotypes, but exhibited reduced intestinal proliferation and impaired differentiation of secretory lineage cells. In a murine model of colorectal cancer, Usp28 deletion resulted in fewer intestinal tumors, and importantly, in established tumors, Usp28 deletion reduced tumor size and dramatically increased lifespan. Moreover, we identified Usp28 as a c-MYC target gene highly expressed in murine and human intestinal cancers, which indicates that USP28 and c-MYC form a positive feedback loop that maintains high c-MYC protein levels in tumors. Usp28 deficiency promoted tumor cell differentiation accompanied by decreased proliferation, which suggests that USP28 acts similarly in intestinal homeostasis and colorectal cancer models. Hence, inhibition of the enzymatic activity of USP28 may be a potential target for cancer therapy.