Objectives To investigate the effect of Inonotus obliquus extract on Crohn's disease and its mechanism by proteomics technology. Methods Crohn's disease (CD) model was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). A total of 48 SD male rats were randomized into control, model, mesalazine (225 mg/kg), and low-dose (200 mg/kg), medium-dose (400 mg/kg), high-dose (800 mg/kg) Inonotus obliquus groups. The disease activity index (DAI) score and the colonic mucosal injury index (CMDI) score were assessed after one week of drug intervention. HE staining was used to observe the histopathological changes in the colon, and ELISA was used to detect the levels of IL-1β, IL-6, and TNF-α in the blood. Proteins were extracted from the colonic tissues of the control group, model group, and Inonotus obliquus extract high-dose group, and bioinformatics analysis was performed for the proteins identified by quantitative proteomics. Finally, WB and RT-PCR were employed to verify the key proteins. Results The Inonotus obliquus extract improved the symptoms of colitis induced by TNBS in rat, and decrease the levels of IL-1β, IL-6, and TNF-α in the blood. Proteomic tests showed that there were 199 differentially expressed proteins (DEPs) between the Inonotus obliquus extract high-dose group and the model group, of which 63 DEPs were related to CD. Bioinformatics analysis showed that these 63 DEPs were mainly involved in NOD-like receptor signaling pathway, calcium signaling pathway, necroptosis, and other pathways. Consistent with proteomic results, expressions of Vdac1 and Trpv2 were confirmed by WB and RT-PCR in colon tissue. Conclusion Inonotus obliquus extract may regulate NOD-like receptor signaling pathway, calcium signaling pathway, and necroptosis by interfering with the expression of Vdac1 and Trpv2, so as to achieve the effect of treating CD.