Abstract: This study aimed to investigate the effects of the polysaccharide of Atractylodes macrocephala Koidz (PAMK) on cyclophosphamide (CTX)-induced renal injury in mice and its potential underlying mechanisms. One hundred male C57BL/6 mice, aged 42~43 days, were randomly divided into four groups, with five repetitions in each group and five mice in each repetition. The PAMK group and PAMK+CTX group were orally administered 200 mg/kg PAMK once daily, while the control group and CTX group were given an equivalent amount of saline. From days 25~27 of the experiment, the CTX group and PAMK+CTX group were intraperitoneally injected with 100 mg/kg CTX once daily, while the control group and PAMK group were injected with an equivalent amount of saline. On day 35 of the experiment, the kidneys were collected for histological observation, oxidative stress detection, and transcriptome sequencing. The results showed that compared with the CTX group, the renal injury in the PAMK+CTX group was alleviated. The content of malondialdehyde (MDA) in the kidneys was significantly decreased (P<0.05), while the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC) were significantly increased (P<0.05). To further explore the regulatory mechanisms of PAMK in alleviating CTX-induced renal injury, transcriptome sequencing of the kidneys was performed. The results showed that compared with the control group, 493 differentially expressed genes (DEGs) were identified in the CTX group vs control group comparison, and 333 DEGs were identified in the CTX group vs PAMK+CTX group comparison. Functional enrichment analysis of the DEGs in both groups revealed significant enrichment in signaling pathways related to arachidonic acid metabolism. The expression levels of arachidonic acid pathway-related genes were examined, and it was found that compared with the control group, the mRNA expression levels of Cyp2b9, PTGS1, NF-κB and Mfsd2a were significantly increased in the CTX group (P<0.05). On the other hand, in the PAMK+CTX group, the expression levels of Cyp2c65 and BCL6 were significantly increased (P<0.05), while the expression levels of Cyp2b9, PTGS1and Mfsd2a were significantly decreased (P<0.05). In conclusion, PAMK may alleviate oxidative stress in the kidneys of mice and reduce CTX-induced renal injury through the arachidonic acid metabolism pathway.