Background and Purpose: Diabetic nephropathy (DN) closely relates to morphological and functional changes in podocytes, and anaerobic glycolysis represents the predominant energy source of podocytes. However, it is unknown whether lactate accumulation in chronic high glucose caused epithelial-mesenchymal transition (EMT) of podocytes through lactate-derived histone lactylation. Experimental Approach: We examined biomarkers of podocytes and mesenchymal cells as well as lactylation of histone lysine residues (HKla) in mouse MPC cells cultured with high glucose (HG) or lactate (LA). Moreover, these indices were observed in MPCs after HG co-culture with multiple interventions of lactate levels, and differently expressed genes (DEGs) were screened using RNA sequencing. Finally, renal pathological characteristics and histone lactylation were investigated in diabetic mice with lactate-lowering treatments. Key Results: Both HG and LA decreased nephrin levels while increased collagen IV levels in MPCs, and HG and LA stimulation synchronously elevated HKla levels. However, co-treatment with oxamate or dichloroacetate reducing lactate levels alleviated decreases in nephrin and ZO-1 levels and increases in collagen IV and α smooth muscle actin levels as well as HKla levels in HG-cultured MPCs, but co-treatment with rotenone diversely affected these indices. RNA sequencing found eleven DEGs in HG-cultured MPCs after oxamate or dichloroacetate intervention and qPCR experiments validated four of them. Importantly, oxamate or dichloroacetate treatment attenuated renal functions, EMT, and histone lactylation in kidney of diabetic mice. Conclusion and Implications: This study clarified that lactate mediated chronic high glucose-caused podocyte EMT through lactate-induced histone lactylation, and then promoted the pathological process of DN.