Ectopic fat deposition (EFD) in the kidney has been shown to play a causal role in diabetic nephropathy; however, the mechanism underlying EFD remains elusive. By transcriptome analysis, we found decreased expression levels of disulfide-bond A oxidoreductase-like protein (DsbA-L) in the kidneys of diabetic mice (induced by high-fat diet plus Streptozotocin) compared with control mice. Increased expression of adipocyte differentiation-related protein and abnormal levels of collagen I, fibronectin, and phosphorylated 5'AMP-activated kinase (p-AMPK), adipose triglyceride lipase (p-ATGL), and HMG-CoA reductase (p-HMGCR) were also observed in diabetic mice. These alterations were accompanied by deposition of lipid droplets in the kidney, and were more pronounced in diabetic DsbA-L knockout mice. In vitro, overexpression of DsbA-L ameliorated high glucose-induced intracellular lipid droplet deposition in a human proximal tubular cell line, and DsbA-L siRNA aggravated lipid droplet deposition and reduced the levels of p-AMPK, p-ATGL, and p-HMGCR. High glucose and palmitic acid treatment enhanced the expression of interleukin-1β and interleukin-18; these enhancements were further increased after treatment with DsbA-L siRNA but alleviated by co-treatment with an AMPK activator. In kidney biopsy tissue from patients with diabetic nephropathy, DsbA-L expression was negatively correlated with EFD and tubular damage. Collectively, these results suggest that DsbA-L has a protective role against EFD and lipid-related kidney damage in diabetic nephropathy. Activation of the AMPK pathway is a potential mechanism underlying DsbA-L action in the kidney.