Abstract Supplementation of maturation media with antioxidant (bulk form) improves oocyte maturation. However, the influence of adding antioxidant (nano-particles) on oocyte maturation is not well known. We aimed to evaluate the effect of selenium nano-particles (SeNP) and bulk selenium (Se) on buffalo oocytes maturation, in terms nuclear maturation and molecular level. Oocytes were distributed into four groups; 1st group was control, 2nd group was supplied with Se (10 ng/ml), 3rd and 4th groups were supplied with 1 μg/ml SeNP (67 nm), and SeNP (40 nm), respectively. Matured oocytes were fixed and stained to determine nuclear maturation. Oocytes and COC after IVM were stored at − 80 °C, for RNA isolation and qRT-PCR for selected genes. The Se and seNP (40 nm) had a positive effect on oocytes nuclear maturation rates. Apoptosis-related cysteine peptidase (CASP3) was reduced in all supplemented groups. Anti-Mullerian hormone (AMH) was up-regulated in oocytes supplemented with SeNP (40 nm). In COC, AMH increased in group supplemented with SeNP (67 nm). In oocytes, phospholipase A2 group III (PLA2G3) decreased in all supplemented groups. While in COC, PLA2G3increased in group supplied with Se. In COC, luteinizing hormone/choriogonadotropin receptor (LHCGR) increased in groups supplied with Se or SeNP (40 nm).Glutathione peroxidase 4 (GPX4) increased in all supplemented groups, in oocytes and COC. In oocytes, superoxide dismutase (SOD) was up-regulated in supplemented groups {Se and SeNP (67 nm)}.The DNA methyltransferase (DNMT) in oocytes was reduced in supplemented groups. In oocytes, the POU class 5 homeobox 1 (OCT4) increased in all supplemented groups. In COC, the OCT4 was over-expressed in group supplemented with SeNP (40 nm). Selenium supplementation in bulk or nano-particle improved in vitro buffalo oocytes maturation, viaup-regulation of antioxidant defense and development competence genes. SeNP (smaller size, 40 nm) induced higher expression of antioxidant gene.