The compound ganoderma lucidum spore powder (GLSP) has emerged as an anti-inflammatory and anti-oxidative regulator. In this study, we explored the roles of GLSP against dextran sulfate sodium (DSS)-induced mouse colitis that can mimic human inflammatory bowel disease (IBD). GLSP was administered by oral gavage at a dosage of 150 mg/kg/day to the acute colitis mice induced by DSS. The DSS-induced mouse weight loss, colonic shortening, diarrhea and bloody stool were observably alleviated after GLSP treatment. The lesion of macroscopic and microscopic signs of the disease was reduced significantly and DSS-induced gut barrier dysfunction was restored via increasing the level of claudin-1, ZO1, Occu, and ZO2 with GLSP. Meanwhile, the levels of IL-6, TNF-α, IL-1β, and IL-18 in the colon were reduced in the GLSP-treated groups. In addition, phosphorylation of the MAPKs ERK1/2, p38, and AKT was suppressed after GLSP treatment. All these results demonstrated that GLSP owned a protective effect on DSS-induced colitis by inhibition of MAPK pathway, which provides a promising therapeutic approach for the treatment of colitis.