The in situ metabolic profiling of the kidney is crucial to investigate the complex metabolic reprogramming underlying diabetic kidney disease (DKD) and to allow exploration of potential metabolic targets to improve kidney function. However, as the kidney is a highly heterogeneous organ, traditional metabolomic methods based on bulk analysis that produce an averaged measurement are inadequate. Herein, we employed an in situ metabolomics approach to discover alternations of DKD-associated metabolites and metabolic pathways. A series of histology-specific metabolic disturbances were discovered in situ using airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI–MSI). In combination with integrated metabolomics analysis, five dysfunctional metabolic pathways were identified and located in the kidneys of type-2 DKD mice simultaneously for the first time, including taurine metabolism, arginine and proline metabolism, histidine metabolism, biosynthesis of unsaturated fatty acids, and fatty acid degradation pathways. As crucial nodes of metabolic pathways, five dysregulated rate-limiting enzymes related to altered metabolic pathways were further identified. These findings reveal alternations from metabolites to enzymes at the molecular level in the progression of DKD and provide insights into DKD-associated metabolic reprogramming.