Gut microbiota-derived metabolites play important roles in health and disease. D-amino acids and their L-forms are metabolites of gut microbiota with distinct functions. In this study, we show the pathophysiologic role of D-amino acids in association with gut microbiota in humans and mice with acute kidney injury (AKI). In a mouse kidney ischemia/reperfusion model, the gut microbiota protected against tubular injury. AKI-induced gut dysbiosis contributed to the altered metabolism of D-amino acids. Among the D-amino acids, only D-serine was detectable in the kidney. In injured kidneys, the activity of D-amino acid oxidase was decreased. Conversely, the activity of serine racemase was increased. The oral administration of D-serine mitigated the kidney injury in B6 mice and D-serine-depleted mice. D-serine suppressed hypoxia-induced tubular damage and promoted posthypoxic tubular cell proliferation. Finally, the D-serine levels in circulation were significantly correlated with the decrease in kidney function in AKI patients. These results demonstrate the renoprotective effects of gut-derived D-serine in AKI, shed light on the interactions between the gut microbiota and the kidney in both health and AKI, and highlight D-serine as a potential new therapeutic target and biomarker for AKI.