Abnormal lipid/lipoprotein metabolism induced by obesity may affect spermatogenesis by inhibiting testosterone synthesis in Leydig cells. It is crucial to determine which components of lipoproteins inhibit testosterone synthesis. Circulating oxidized low-density lipoprotein (oxLDL), the oxidized form of LDL, has been reported to be an independent risk factor for decreased serum testosterone levels. However, whether oxLDL has a damaging effect on Leydig cell function and the detailed mechanisms have been rarely studied. This study first showed the specific localization of oxLDL and mitochondrial structural damage in testicular Leydig cells of high-fat diet-fed mice in vivo. We also found that oxLDL reduced the mitochondrial membrane potential (MMP) by disrupting electron transport chain and inhibited testosterone synthesis-related proteins and enzymes (StAR, P450scc, and 3β‑HSD), which ultimately led to mitochondrial dysfunction and decreased testosterone synthesis in Leydig cells. Further experiments demonstrated that oxLDL promoted lipid uptake and mitochondrial dysfunction by inducing CD36 transcription. Meanwhile, oxLDL facilitated COX2 expression through the p38 MAPK signaling pathway in Leydig cells. Blockade of COX-2 attenuated the oxLDL-induced decrease in StAR and P450scc. Our clinical results clarified that the increased serum oxLDL level was associated with a decline in circulating testosterone levels. Our findings amplify the damaging effects of oxLDL and provide the first evidence that oxLDL is a novel metabolic biomarker of male-acquired hypogonadism caused by abnormal lipid metabolism.