High vitamin D3 receptor and vitamin D3 metabolizing enzyme expression during spermatogenesis reflects the importance of vitamin D in fertility. This project aimed to study the effects of vitamin D3 supplementation on semen parameters and the oxidative system of rams. Twenty sexually active, mature rams were randomly selected. Rams were evenly (based on their age and live weight) categorized into four groups: control group (CG), low-dose vitamin D3 treatment group (LDG), high-dose vitamin D3 treatment group (HDG), and normal group (NG). The CG, LDG, and HDG were kept in a dark place for three months, and the vitamin D3 supplement was removed from the diet to cause vitamin D3 deficiency. The NG was placed in free sunlight, and a vitamin D3 supplement was added to their diet; thus, the vitamin D3 serum levels were normal in this group. After vitamin D3 deficiency (< 75 nmol/L vitamin D in the serum), rams received 2000IU/kg vitamin D3 in the LDG. Rams in the HDG received 20000IU/kg of vitamin D3. The rams in the CG group did not receive vitamin D3 supplementation. Sperm collection was performed three times in the morning for three consecutive months. The results showed a relationship between vitamin D3 serum level, semen parameters, and oxidative stress indices. After the treatment, vitamin D3 serum levels in LDG (128.4±17.3 nmol/L) and HDG (119.3±10.6 nmol/L) were statistically (P<0.05) different from CG (42.9±3.6 nmol/L). Treatment with both doses of vitamin D3 significantly improved sperm parameters (P < 0.05). Sperm viability (95±6% in LDG, 95±4% in HDG, and 61±1.1% in CG), sperm PMI (85±5% in LDG and 84±5% in HDG, whereas 51±8% in CG), sperm morphology (9±3% in LDG and 10±2% in HDG in contrast with 28±6% in CG), and DNA fragmentation index (10±3% in LDG, 11±1% in HDG, and 31±7% in CG) were statistically different (P < 0.05). Treatment with both doses of vitamin D3 substantially (P<0.05) declined malondialdehyde concentration (1.2±0.4 μmol/L in LDG and 1±0.3 μmol/L in HDG in contrast with 9.7±0.9 μmol/L in CG) and total oxidant status level (9.9±3.8 μmol H2O2 equiv/L in LDG and 10±3.6 μmol H2O2 equiv/L in HDG whereas 77±10.6 μmol H2O2 equiv/L in CG). However, total antioxidant capacity concentration (4.17±0.61 μmol fe2+/L in LDG and 4.86±0.62 μmol fe2+/L in HDG while 1.02±0.29 μmol fe2+/L in CG) and total thiol concentration (231.9 ± 23.8 μmol/mg protein in LDG and 250.6 ± 25.9 μmol/mg protein in HDG in contrast with 31.5 ± 11.2 μmol/mg protein in CG) statistically (P<0.05) increase in LDG and HDG. In conclusion, vitamin D3 supplementation improved semen quality in rams by its vitamin D3 receptor action and oxidative stress reduction. Keywords: vitamin D3, oxidative stress,

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