The mechanisms via which inflammatory macrophages mediate intestinal inflammation are not completely understood. Herein, using merged analysis of RNA sequencing and mass spectrometry-based quantitative proteomics, we detected differences between proteomic and transcriptomic data in activated macrophages. Dipeptidase-2 (DPEP2), a member of the DPEP family, was highly expressed and then downregulated sharply at the protein level but not at the mRNA level in macrophages in response to inflammatory stimulation. Suppression of DPEP2 not only enhanced macrophage-mediated intestinal inflammation in vivo but also promoted the transduction of inflammatory pathways in macrophages in vitro. Mechanistically, overexpressed DPEP2 inhibited the transduction of inflammatory signals by resisting MAK3K7 in inactivated macrophages, whereas DPEP2 degradation by activated Trim32 resulted in strong activation of NF-κB and p38 MAPK signaling via the release of MAK3K7 in proinflammatory macrophages during the development of intestinal inflammation. The Trim32-DPEP2 axis accumulates the potential energy of inflammation in macrophages. These results identify DPEP2 as a key regulator of macrophage-mediated intestinal inflammation. Thus, the Trim32-DPEP2 axis may be a potential therapeutic target for the treatment of intestinal inflammation.