Colitis can significantly impact daily life. This study utilized DSS to induce acute colitis in mice and examined the regulatory effect of arabinogalactan (AG). The findings demonstrated that AG intake effectively alleviated the phenotype of DSS-induced colitis in mice and protected against small intestine damage. Furthermore, AG suppressed the secretion of pro-inflammatory factors TNF-α and IL-1β, while promoting the secretion of anti-inflammatory factor IL-10. It also inhibited the secretion of LPS in serum and MPO in colon tissue. Additionally, AG regulated the NF-κB/MAPK/PPARγ signaling pathway and inhibited the NLRP3 inflammasome signaling pathway, thereby ameliorating DSS-induced colitis inflammation in mice. AG also influenced the metabolism of short-chain fatty acids, particularly butyrate, in the intestinal tract of mice. Moreover, AG modulated and enhanced the composition of intestinal flora in mice with colitis, increasing the diversity of dominant flora and promoting the growth of beneficial bacteria. These results highlight the protective effects of arabinogalactan against colitis and its potential applications in the food industry.