The Agrocybe cylindracea polysaccharides (ACP) have been shown to possess various health benefits. However, the anti-inflammatory effects of ACP and the mechanisms underlying these effects remain poorly understood. In this study, we delved into the potential of ACP in mitigating colitis induced by dextrose sodium sulfate (DSS) in C57BL/6J mice and explored the associated mechanisms. ACP demonstrated notable anti-inflammatory effects in the intestines, as evidenced by the restoration of various indicators such as increased body weight and colon length, decreased disease activity index (DAI) score, and alleviation of pathological damage in the colon. Additionally, ACP upregulated the expression of ZO-1, Occludin, and Muc2, suggesting a restoration of the mechanical and mucus barriers of the intestinal mucosa. Furthermore, ACP facilitated the polarization of M2 macrophages and preserved intestinal immune balance. The impact of ACP on the biological barrier was further scrutinized. ACP ameliorated disruptions in the gut microbiota, particularly by enhancing the relative abundance of bacteria that produce short-chain fatty acids (Anaerostipes, Romboutsia, and Butyricicoccus). Remarkably, the ameliorative effects of ACP on colitis were compromised upon the removal of the microbial community, but were restored post fecal microbiota transplantation (FMT), indicating that the efficacy of ACP in alleviating colitis is dependent on the gut microbiota. Intriguingly, the regulatory influence of ACP on macrophages also hinged on the gut microbiota. In essence, ACP exhibits the potential to modulate the composition of the gut microbiota, fostering the growth of beneficial bacteria, which in turn promotes the polarization of colonic M2 macrophages and facilitates the repair of the intestinal mucosal barrier in DSS-induced colitis. These findings underscore the capacity of ACP to enhance the intestinal mucosal barrier and ameliorate colitis by modulating the gut microbiota-macrophage axis, suggesting that ACP could serve as a functional food supplement for preserving intestinal homeostasis and preventing colitis.