Objective 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, Inonotus obliquus low-dose (200 mg/kg), medium-dose (400 mg/kg), high-dose (800 mg/kg) groups, and positive control group (mesalazine, 225 mg/kg). 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 serum. Proteins were extracted from the colonic tissues of the control group, model group, and Inonotus obliquus high-dose group, and bioinformatics analysis was performed for the proteins identified by quantitative proteomics. Finally, Western blot and RT-qPCR were employed to verify the key proteins. Results Compared with the model group, the DAI, CMDI and HE staining scores were significantly decreased in the medium and high dose groups (P<0.05 or P<0.01), as well as the levels of inflammatory factors IL-1β, IL-6 and TNF-α in serum (P<0.05 or P<0.01). Proteomic tests showed that there were 199 differentially expressed proteins (DEPs) between the Inonotus obliquus 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 result, expressions of Vdac1 and Trpv2 were confirmed by Western blot and RT-qPCR in colon tissue. Conclusions 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.