High vitamin D-3 receptor and vitamin D-3 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 D-3 treatment group (LDG), high-dose vitamin D-3 treatment group (HDG), and normal group (NG). The CG, LDG, and HDG were kept in a dark place for three months, and the vitamin D-3 supplement was removed from the diet to cause vitamin D-3 deficiency. The NG was placed in free sunlight, and a vitamin D-3 supplement was added to their diet; thus, the vitamin D-3 serum levels were normal in this group. After vitamin D-3 deficiency (< 75 nmol/L vitamin D in the serum), rams received 2000IU/kg vitamin D-3 in the LDG. Rams in the HDG received 20000IU/kg of vitamin D-3. The rams in the CG group did not receive vitamin D-3 supplementation. Sperm collection was performed three times in the morning for three consecutive months. The results showed a relationship between vitamin D-3 serum level, semen parameters, and oxidative stress indices. After the treatment, vitamin D-3 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 D-3 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 D-3 substantially (P < 0.05) declined malondialdehyde concentration (1.2 +/- 0.4 <mu>mol/L in LDG and 1 +/- 0.3 mu mol/L in HDG in contrast with 9.7 +/- 0.9 mu mol/L in CG) and total oxidant status level (9.9 +/- 3.8 mu mol H2O2 equiv/L in LDG and 10 +/- 3.6 mu mol H2O2 equiv/L in HDG whereas 77 +/- 10.6 mu mol H2O2 equiv/L in CG). However, total antioxidant capacity concentration (4.17 +/- 0.61 mu mol fe(2+)/L in LDG and 4.86 +/- 0.62 mu mol fe(2+)/L in HDG while 1.0(2 +/-)0.29 mu mol fe(2+)/L in CG) and total thiol concentration (231.9 +/- 23.8 mu mol/mg protein in LDG and 250.6 +/- 25.9 mu mol/mg protein in HDG in contrast with 31.5 +/- 11.2 mu mol/mg protein in CG) statistically (P < 0.05) increase in LDG and HDG. In conclusion, vitamin D-3 supplementation improved semen quality in rams by its vitamin D-3 receptor action and oxidative stress reduction.