Strengthening the gut epithelial barrier is a potential strategy for management of gut microbiota-associated illnesses. Here, we demonstrate that dual-specificity phosphatase 6 (Dusp6) knockout enhances baseline colon barrier integrity and ameliorates dextran sulfate sodium (DSS)-induced colonic injury. DUSP6 mutation in Caco-2 cells enhances the epithelial feature and increases mitochondrial oxygen consumption, accompanied by altered glucose metabolism and decreased glycolysis. We find that Dusp6-knockout mice are more resistant to DSS-induced dysbiosis, and the cohousing and fecal microbiota transplantation experiments show that the gut/fecal microbiota derived from Dusp6-knockout mice also confers protection against colitis. Further culturomics and mono-colonialization experiments show that one gut microbiota member in the genus Duncaniella confers host protection from DSS-induced injury. We identify Dusp6 deficiency as beneficial for shaping the gut microbiota eubiosis necessary to protect against gut barrier-related diseases.