This study explored the potential therapeutic effect and possible mechanism of Atractylodes macrocephala Koidz. Polysaccharide (AP) on pirarubicin chemotherapy-induced depression (CID) in breast cancer mice. This study utilized a variety of techniques to explore the potential of AP in mitigating behavioral abnormalities and elucidate the role of gut microbiota regulation in its therapeutic effects on chemotherapy in breast cancer mice. These included a chemotherapy mouse model, behavioral assessments, histological analysis using hematoxylin and eosin staining, ultrastructural examination, enzyme-linked immunosorbent assays, 16S rDNA sequencing, metabolomic profiling, Western blot analysis, and a pseudo-germ-free animal model. Oral administration of AP significantly improved depression-like behaviors in breast cancer chemotherapy mice while also reducing neuronal damage and inflammation in the hippocampus. AP prevented ferroptosis of intestinal tissues caused by chemotherapy and had a repairing effect on the intestinal barrier damage of chemotherapy-induced mice. Additionally, AP enhanced gut microbiota composition and altered intestinal metabolites in chemotherapy-treated mice. It notably decreased the abundance of certain microbes, such as Bacteroidaceae, Lachnospiraceae, Oscillospiraceae, and Clostridium, while significantly increasing the abundance of Alistipes. Moreover, AP efficiently modulated intestinal metabolites, including glycocholic acid, L-Phenylalanine, and palmitoylcarnitine. More importantly, depletion of gut microbiota through antibiotics diminished the effectiveness of AP. Our results suggest that AP alleviates depression-like behaviors in chemotherapy-treated mice by regulating the gut microbiota and microbial metabolism, as well as suppressing ferroptosis in intestinal tissues.