Oxidative stress plays a key role in the pathogenesis of diabetic nephropathy (DN). The anti-aging protein Klotho has been demonstrated to have antioxidant capacity. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a central transcription factor regulating antioxidant responses. The present study aimed to explore the effects of Klotho on DN and the underlying mechanisms related to Nrf2. Low glucose (LG) or high glucose (HG) medium-cultured podocytes and diabetic db/db mice were overexpressed with Klotho via adenoviral transfer to evaluate the effects of Klotho on Nrf2 signaling, oxidative stress, podocyte apoptosis, and renal function and histopathology. Klotho overexpression significantly induced the expression and activation of Nrf2 as well as its downstream targets SOD2 and NQO1 in podocytes. Moreover, Klotho overexpression inhibited HG-induced oxidative stress and apoptosis in podocytes. Co-treatment with Nrf2 inhibitor trigonelline prevented Klotho-induced expression of SOD2 and NQO1, and abolished Klotho-conferred antioxidant and anti-apoptotic effects. In db/db mice, Klotho overexpression also activated Nrf2 signaling, and suppressed diabetes-induced oxidative stress and podocyte apoptosis, which were accompanied by improved renal function and decreased glomerulosclerosis. Our data highlight a novel Nrf2-mediated antioxidant mechanism underlying the protective effects of Klotho in podocytes and indicate the therapeutic potential of targeting Klotho to activate Nrf2 in DN.