The current study was conducted to test the possible ameliorative role of selenium nanoparticles (Se-NPs) against oxidative damage of Leyding cells induced by di-n-butyl phthalate (DBP) in pre-pubertal male rat offspring. Forty-two pregnant female rats treated from gestation day (GD) 12 to postnatal day (PND) 14 day with two doses of Se-NPs (0.2 and 0.5 mg/kg/d) against developmental testicular toxicity induced by DBP (500 mg/kg/d). At PND 25 serum and testes of offspring were collected. Serum LH, the Leydig cells performance [total serum testosterone, LH and testosterone (LH/T) ratio, relative gene expression of insulin-like growth factor-3 (INSL3) and mineralocorticoid receptor (MR)], oxidative stress biomarker malondialdehyde (MDA) and antioxidant machinery [reduced glutathione (GSH), and the relative gene expression of antioxidant enzymes: superoxide dismutase (SOD), glutathione peroxidase (GPx)] were estimated in all groups. The obtained results revealed that maternal exposure to DBP significantly reduced total serum testosterone level, relative mRNA expression of INSL3 and MR genes with observed testicular damage revealed by increasing MDA and depressed levels of GSH and antioxidant enzymes. The histopathological changes include necrosis and desquamation of spermatogoneal cells. Co-administration of Se-NPs high dose along with DBP significantly increased serum testosterone, improved LH/T ratio and the relative mRNA expression of INSL3 and MR genes, decreased the level of MDA, and also improved all the antioxidant enzymes expression levels. In conclusion, Se-NPs could be a potent maternal prophylactic agent against the reduced total serum testosterone level and oxidative damage of Leydig cells induced by DBP via reducing the lipid peroxidation (LPO) and enhancing the antioxidant state in pre-pubertal male rat offspring.