It is widely believed that grape seed proanthocyanidin extract (GSPE) exerts antioxidant and anti-inflammatory effects. Dietary supplementation with GSPE has been reported to alleviate colitis signs in mice, but the mechanisms involved require further exploration. The present study investigated how the oral administration of GSPE ameliorates colitis signs and reduces colitis-associated inflammation. C57BL/6 mice were treated with GSPE for 21 days. During the final 7 days of treatment, the mice were administered dextran sulfate sodium (DSS) dissolved in drinking water to induce experimental colitis. We found that GSPE treatment improved DSS-induced colitis, which was evidenced by decreases in disease activity index (DAI) scores, pathological scores, and oxidative stress and increases in zonula occludens-1 (ZO-1), occludin, and claudin-1 mRNA levels of colon tissue. Notably, the proinflammatory cytokines TNF-α and IL-1β were significantly downregulated as a result of GSPE treatment in colon tissues. GSPE treatment also reduced NLR family pyrin domain-containing 3 (NLRP3) inflammasome mRNA levels of colon tissue. Furthermore, an analysis of 16S rRNA sequences showed that GSPE rebalanced the DSS-damaged gut microbiota, including reducing Bacteroidetes, Dubosiella, and Veillonella, increasing Verrucomicrobia and Akkermansia, and elevating the Firmicutes to Bacteroidetes ratio. In conclusion, GSPE supplementation alleviates DSS-induced colitis by modulating inflammatory cytokines and oxidation stress, maintaining the intestinal barrier, and improving the microbial community. These results indicate that GSPE might be a new dietary strategy for the treatment of ulcerative colitis.