Macrophages drive the pathological process of inflammatory bowel diseases (IBD) mostly by secreting proinflammatory cytokines, such as Tnf-α. Recent studies have indicated the association between epigenetic modifications and macrophage functions. However, epigenetic mechanisms regulating macrophages’ functional involvement in IBD remain unknown. In this study, we investigated whether the epigenetic regulator Uhrf1 plays a role in innate immunity by functionally regulating macrophages in intestines. We employed two transgenic strains of mice (one with Uhrf1 deficiency in macrophages [Uhrf1 fl/fl Lyz2-Cre mice] and the other with the two mutations at Uhrf1’s DNA methylation regulatory site [Uhrf1 YP187/188AA mice]) to assess their susceptibility to dextran sodium sulfate–induced colitis. We examined the cytokines derived from Uhrf1 fl/fl Lyz2-Cre and Uhrf1 YP187/188AA macrophages in response to LPS stimulation. We also analyzed the effects of proinflammatory cytokines on Uhrf1 expression in macrophages. The data demonstrated that Uhrf1 deficiency and Uhrf1 YP187/188AA mutation resulted in severe colitis in the dextran sodium sulfate–treated mice. In vitro analysis revealed the hypomethylation of Tnf-α promoter and the increased Tnf-α expression in Uhrf1 fl/fl Lyz2-Cre and Uhrf1 YP187/188AA macrophages in response to LPS stimulation, and anti–Tnf-α therapy implied the key role of Tnf-α to the aggravated colitis in Uhrf1-deficient mice. Exogenous Tnf-α destabilized Uhrf1 protein through ubiquitination-mediated protein degradation, triggering macrophage activation. In conclusion, we identified that Uhrf1-mediated DNA methylation controls Tnf-α expression of macrophages in the experimental colitis resembling IBD. The epigenetic mechanisms that activate macrophages may provide new therapeutic targets for IBD treatment.