Diabetic kidney disease (DKD) progression is predominantly attributed to podocyte injury. However, the underlying mechanisms remain inadequately understood. DKD patients exhibit elevated lactate concentrations due to impaired glucose metabolism. Lactate serves as a substrate for a post-translational modification known as lactylation, which is integral to numerous cellular processes. The study aimed to explore the potential role of lactylation in DKD podocyte injury and to elucidate its underlying mechanisms. The results showed that lactate concentrations were elevated in the plasma of DKD patients, as well as in the serum and renal tissue of diabetic mice. High glucose (HG) resulted in increased lactate accumulation in podocytes, exacerbated podocyte injury and promoted protein lactylation. Protein modification omics demonstrated that LARS1 lactylation was enhanced, specifically at the K970 site, which further aggravated podocyte injury by inhibiting autophagy and promoting apoptosis. In vivo transfection with LARS1 siRNA significantly improved renal function and mitigated podocyte injury. Our study identified a novel mechanism linking LARS1 lactylation activity to compromised podocyte autophagy, heightened podocyte injury, and the onset of proteinuria within DKD.