Dehydroepiandrosterone (DHEA) possess anti-inflammatory, anti-oxidant and immune-regulating function in animals and humans, but there is not enough information about the mechanisms underlying its beneficial effects. The present study investigated the effect and mechanism of DHEA in dextran sulfate sodium (DSS)-induced colitis mice. The findings showed that DHEA relieved the decreasing of body weight, the increasing of disease activity index, the enhancing of spleen weight, the shortening of colon length and the rising of myeloperoxidase activity; meanwhile, histopathological analysis showed that DHEA maintained a relatively intact structure of colon in DSS-induced colitis mice. DHEA decreased the malondialdehyde content, superoxide dismutase activity and inducible nitric oxide synthase protein level; meanwhile, DHEA also inhibited the secretion of tumor necrosis factor-α, interleukin-1β and interleukin-6 in DSS-induced colitis mice. Importantly, our results showed that DHEA blocked the activation of nuclear factor-kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) pathways; and it inhibited the Nod-like receptor protein 3 inflammasome activation in DSS-induced colitis mice. Furthermore, DHEA markedly promoted the intestinal barrier function by up-regulation zonula occludens-1 expression level. The 16S rDNA gene sequencing demonstrated that DHEA decreased the Pseudomonas abundance in DSS-induced colitis mice. In conclusion, our data demonstrated that DHEA reduces oxidative damage through regulating antioxidant enzyme activity; inhibits pro-inflammatory cytokines production by blocking the activation of p38 MAPK and NF-κB signal pathway; protects colon barrier integrity via increasing tight junction protein expression and modulating gut microbiota taxa; all that finally alleviates DSS-induced experimental colitis in mice.