Obesity has been recognized as a major risk factor for chronic kidney disease, but the underlying mechanism remains elusive. Here, we investigated the mechanism whereby long-term high-fat diet (HFD) feeding induces renal injury in mice. The C57BL/6 mice fed HFD for 16 weeks developed obesity, diabetes, and kidney dysfunction manifested by albuminuria and blood accumulation of BUN and creatinine. The HFD-fed kidney showed marked glomerular and tubular injuries, including prominent defects in the glomerular filtration barrier and increased tubular cell apoptosis. Mechanistically, HFD feeding markedly increased triglyceride and cholesterol contents in the kidney and activated lipogenic pathways for cholesterol and triglyceride synthesis. HFD feeding also increased oxidative stress and induced mitochondrial fission in tubular cells, thereby activating the pro-apoptotic pathway. In HK-2 and mesangial cell cultures, high glucose, fatty acid, and TNF-α combination was able to activate the lipogenic pathways, increase oxidative stress, promote mitochondrial fission, and activate the pro-apoptotic pathway, all of which could be attenuated by an inhibitor that depleted reactive oxygen species. Taken together, these observations suggest that long-term HFD feeding causes kidney injury at least in part as a result of tissue lipid accumulation, increased oxidative stress, and mitochondrial dysfunction, which promote excess programmed cell death.