As the prevalence of inflammatory bowel diseases (IBD) rises, the etiology of IBD draws increasing attention. Glucoraphanin (GRP), enriched in cruciferous vegetables, is a precursor of sulforaphane, known to have anti-inflammatory and antioxidative effects. We hypothesized that dietary GRP supplementation can prevent mitochondrial dysfunction and oxidative stress in an acute colitis mouse model induced by dextran sulfate sodium (DSS). Eight-week-old mice were fed a regular rodent diet either supplemented with or without GRP. After 4 weeks of dietary treatments, half of the mice within each dietary group were subjected to 2.5% DSS treatment to induce colitis. Dietary GRP decreased DSS-induced body weight loss, disease activity index, and colon shortening. Glucoraphanin supplementation protected the colonic histological structure, suppressed inflammatory cytokines, interleukin (IL)-1β, IL-18, and tumor necrosis factor-α (TNF-α), and reduced macrophage infiltration in colonic tissues. Consistently, dietary GRP activated AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α, and nuclear factor erythroid 2-related factor 2 (NRF2) pathways in the colonic tissues of DSS-treated mice, which was associated with increased mitochondrial DNA and decreased content of the oxidative product 8-hydroxydeoxyguanosine (8-OHDG), a nucleotide oxidative product of DNA. In conclusion, dietary GRP attenuated mitochondrial dysfunction, inflammatory response, and oxidative stress induced by DSS, suggesting that dietary GRP provides a dietary strategy to alleviate IBD symptoms.