Ubiquitin-specific protease 25 (USP25), a member of the deubiquitination family, plays an important role in protein ubiquitination, degradation, inflammation, and immune regulation. However, the role and mechanism of USP25 in ulcerative colitis (UC) remain unclear. To study the role and mechanism of USP25 in UC, bioinformatics analysis and research are conducted on clinical patients with UC, Usp25 knockout (Usp25-/-) mice, intestinal epithelial cell-specific knockout signal transducer and activator of transcription 3 (Stat3) (Villin-Cre Stat3fl/fl) mice, and human colonic epithelial cells. Results show that the expression of USP25 is decreased in patients with UC and mice with dextran sulfate sodium salt (DSS)-induced colitis and that USP25 deficiency exacerbates UC by destroying the intestinal mucosal barrier, however, overexpression of USP25 can alleviate colitis. Mechanistically, USP25 reduces the degradation of phosphor-STAT3Y705 at lysine 409 by catalyzing K48-linked deubiquitination. Further, this study demonstrates the aggravation of DSS-induced colitis by intestinal epithelial cell-specific knockout Stat3 in mice, while Stat3 overexpression by adeno-associated virus attenuates colitis in DSS-induced Usp25-/- mice. Together, these results showed that USP25 ameliorates UC by regulating the degradation of phosphor-STAT3. Collectively, USP25 is a specific STAT3 regulator that can be targeted in UC.