Patients with intestinal barrier injury are at high risk of developing various infectious complications with high mortality rate. Therefore, the current study aims to identify novel biomarkers and treatment targets for intestinal mucosa repairing to provide potential therapeutic clues for improving functional recovery and prognostic performance after gut injury. We conducted a large-scale screening of multiple transcriptomic and scRNA-seq datasets of patients with inflammatory bowel disease (IBD), and identified 10 potential key regulatory genes of intestinal barrier repairing: AQP8, SULT1A1, HSD17B2, PADI2, SLC26A2, SELENBP1, FAM162A, TNNC2, ACADS, and TST. Interrogation of their expression pattern using a published scRNA-seq dataset revealed that the expression of these markers is restricted to the territory of absorptive cell population in intestinal epithelium. Furthermore, we conducted a clinical study where 11 patients underwent ileostomy after laparoscopic radical resection of colorectal cancer demonstrated that upregulation of post-operational AQP8 and SULT1A1 expression were associated with improved recovery of bowel functions after surgery-induced intestinal injury. In addition, gene expression analyses on intestinal tissues from mouse embryos at the age of embryonic day (E) 14.5, E18.5 and from adult mice exhibited a highly dynamic expression pattern of Aqp8 and Hsd17b2 in intestinal development, implying potential links and functional similarity of the molecular mechanisms in building and repairing intestinal barrier. Together, our work provides new insight into the molecular mechanisms of gut barrier repairing at a multiomics level and suggests multiple confident biomarkers of intestinal barrier healing as potential prognostic markers and therapeutic targets for patients with impaired intestinal barrier functions.