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Crohn’s disease (CD) is a nonspecific inflammatory disease of digestive tract with limited known biomarkers. We aimed to identify potential molecules involved in CD severity. Weighted gene co-expression network and differentially expressed gene analyses were used to explore hub genes associated with inflammation and involved in pathological processes of CD in the GSE83448 cohort. The expression and relationship of genes with clinical characteristics in normal and CD intestinal mucosa were validated in GSE208303 and our research cohorts. Five genes related to disease and inflammation were identified in the clinically significant modules that were differentially expressed in healthy controls and CD patients. Among them, solute carrier family 39 member 5 (SLC39A5) expression was reduced in CD tissues compared with that in normal tissues; low SLC39A5 expression was associated with CD-simplifying endoscopic score and stenosis. Furthermore, we broadened the bioinformatic threshold with qRT-PCR verification and western blot, and found that the expressions of cytochrome P450 family 2 subfamily S member 1 (CYP2S1), fatty acid 2-hydroxylase (FA2H), fatty acid binding protein 1 (FABP1), and Rho family GTPase 3 (RND3) were reduced in CD tissues. Therefore, SLC39A5, CYP2S1, FA2H, FABP1, and RND3 may be novel biomarkers for CD and associated with severity. Highlights SLC39A5 expression was reduced in CD tissues. Low SLC39A5 expression was associated with CD-simplifying endoscopic score and stenosis. CYP2S1, FA2H, FABP1, and RND3 were reduced in CD tissues.