The Atractylodes rhizome, derived from the dried rhizomes of Atractylodes lancea (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz., is a multi-based original medicinal material used in China to treat gastrointestinal diseases. Polysaccharides, a group of active ingredients found in Atractylodes rhizome, exhibit excellent immune regulation, intestinal protection, and liver protection activities. However, the structural differences, immunomodulatory and intestinal protective effects, and mechanisms of polysaccharides derived from Atractylodes lancea or Atractylodes chinensis rhizome remain unclear. This study aims to compare the basic structural differences between Atractylodes lancea rhizome polysaccharide (ALP) and Atractylodes chinensis rhizome polysaccharide (ACP), and elucidate the differences in pharmacological effects and potential mechanisms of the two polysaccharides in immunomodulation and intestinal protection. The results showed that ALP and ACP were composed of fructose, mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose, with minor amount of uronic acid, and exhibited a triple helix structure. Among them, the higher uronic acid (ALP vs ACP: 4.65 ± 0.68% vs 7.38 ± 1.76%) content of ACP indicated stronger acidity compared to ALP. Both ALP and ACP improved phagocytic activity and cytokine secretion levels in RAW 264.7 cells in vitro, with ACP showing a more significant effect. After administering the two kinds of polysaccharides in vivo, mice exhibited improved immunosuppression, significantly restored humoral immune indexes, reduced pathological damage in the jejunum, and increased secretion levels of TNF-α, IL-1β, IL-6, and iNOS. In addition, the number of goblet cells in the jejunum and the expression level of Mucin 2 (MUC2) increased, and the levels of Zonula Occludens-1 (ZO-1), Occludin, Claudin-1, F4/80 antigen, immunoglobulin A (IgA), and secretory immunoglobulin A (sIgA) were significantly higher than those in the model group. Additionally, the activity of ACP is superior to ALP. 16S rDNA sequencing results showed that ACP could not only increase the diversity index of intestinal flora, such as Chao1, observed_features, Pielou_e, Shannon, and Simpson indexes, but also regulate the composition of intestinal flora at the phylum, family and genus levels, reduce the abundance of Firmicutes and Lachnospiraceae_NK4A136_group, and increase the abundance of Bacteroidota, Muribaculaceae, Ruminococcus and Prevotellaceae_UCG-001. Moreover, ACP can also promote the production of short-chain fatty acids (SCFAs) from fermentation products of intestinal flora. These findings indicated that ACP could be used as an effective immunomodulator to improve immunosuppression and intestinal mucosal injury caused by cyclophosphamide (Cy).