Diabetic renal injury was associated with dysbiosis of the gut microbiota and intestinal barrier. Punicalagin (PU) from pomegranates potentially impacts the microbial ecosystem, intestinal barrier, and renal function. Therefore, we hypothesized that PU may improve diabetic renal injury by modulating the gut-kidney axis. The present study evaluated the effect of PU on the gut-kidney axis and kidney function in a diabetic renal injury mouse model induced by a high-fat diet (HFD). Mice were fed a HFD without PU or with at doses of 50 and 100 mg kg-1 d-1 for 8 weeks. Targeted metabolomics by GC-MS and 16S rRNA sequencing were implemented to determine short-chain fatty acids (SCFAs) and microbes. Further RNA sequencing analyses were performed to determine which differentially expressed genes were changed by PU. Compared with the DM model group, PU supplementation improved diabetic renal injury, ameliorated kidney architecture and function, and reshaped gut microbial ecology. Additionally, PU reversed HFD-induced gut barrier dysfunction, promoted cecal SCFA concentrations and inhibited serum lipopolysaccharide (LPS) and diamine oxidase (DAO) levels. Moreover, correlation analysis found that cecal SCFAs were significantly negatively correlated with inflammation-related genes in the kidney. The present results indicated that PU, a promising bioactive polyphenol, successfully improved diabetic renal injury, most likely through the gut-kidney axis.