Background: Obesity is a major health problem which may lead to infertility, vitamin D has anti-inflammatory, anti-oxidant, anti-apoptotic properties and metabolic activities Nano emulsions are very effective delivery system for medicines and nutrients. Nano vitamin D has a greater water solubility and stability compared to the conventional vitamin D. Aim of the work: The study aims to evaluate the potential protective effects of vitamin D3 nanoemulsion intervention against some altered testicular, metabolic and hormonal changes in high-fat diet (HFD)-induced obese rats. Methods: 32 adult male albino rats were equally and randomly divided into four groups: group I (control group on standard diet), group II (untreated HFD), group III (HFD + conventional vitamin D) and group IV (HFD + Nano vitamin D3). Vitamin D3 administration was begun along with the start of introduction of the high fat diet. After eight weeks, body weight (BW) was measured and body mass index (BMI) was calculated then rats were euthanized,. Serum testosterone, insulin and glucose levels were measured; homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. In addition, testicular levels of malondialdhyde (MDA), superoxide dismutase (SOD), reduced glutathione (GSH), TNF-α and IL-6 were measured. The changes in the histopathological aspects of the testis were examined. The testicular weight, testicular coefficient, epidydimal sperm count and motility all were investigated. Results: Nano vitamin D3 treatment showed significant reductions in BMI, fasting serum glucose, insulin resistance, total cholesterol, LDL-c, with significant elevations in serum HDL-c, final testicular weight, testicular coefficient, epidydimal sperm count, sperm motility, and serum testosterone. Moreover, Nano vitamin D3 significantly increased the testicular SOD and GSH with significant decline in the lipid peroxidation contributed to MDA, testicular TNF-α and IL-6 content. Improvement of the metabolic and biochemical measurements induced by Nano vitamin D3 is linked to improvement of testicular histology in obese rats. Interestingly, Nano vitamin D3 has more significant protective effects against HFD-induced testicular dysfunction compared to the conventional vitamin D3. Conclusions: Nano vitamin D3 has a potential protective role in improving the adverse effects of HFD induced obesity on testicular functions, with more prominent protective effects than conventional vitamin D3.