<b><i>Introduction:</i></b> In diabetic nephropathy (DN), mitochondrial dysfunction and leakage of mitochondrial DNA (mtDNA) are caused by the downregulation of superoxide dismutase 2 (SOD2). mtDNA induces the activation of Toll-like receptor (TLR) 9, which is present in macrophages (Mφs), and triggers their activation. <b><i>Methods:</i></b> We orally administered L-carnitine, which exerts protective effects on the mitochondria, to obesity-induced DN (db/db) mice for 8 weeks. We then investigated the effects of L-carnitine on kidney mitochondrial reactive oxygen species (mtROS) production, circulating mtDNA content, and kidney CD11b^high/CD11b^low Mφ functions. <b><i>Results:</i></b> In db/db mice, mtROS production increased in proximal tubular cells and kidney CD11b^low Mφs; both Mφ types showed enhanced TLR9 expression. L-Carnitine treatment suppressed mtROS production in both proximal tubular cells and CD11b^low Mφs (<i>p</i> &#x3c; 0.01), with improved SOD2 expression in the kidney (<i>p</i> &#x3c; 0.01), decreased circulating mtDNA content, and reduced albuminuria. Moreover, it suppressed Mφ infiltration into kidneys and reduced TLR9 expression in Mφs (<i>p</i> &#x3c; 0.01), thereby lowering tumor necrosis factor-α production in CD11b^high Mφs (<i>p</i> &#x3c; 0.05) and ROS production by CD11b^low Mφs (<i>p</i> &#x3c; 0.01). Collectively, these changes alleviated DN symptoms. <b><i>Conclusion:</i></b> The positive effects of L-carnitine on DN suggest its potential as a novel therapeutic agent against obesity-linked DN.