The present study was designed to show the effect of Vitamin D3 on lipid profile and oxidative stress. The present study used 30 adult albino female rats that distributed to following groups (each group consist 6 rats); control group received ad libidium, second group administrated Vitamin D3 (orally, 8.9μg/kg) for eight weeks, third group administrated Vitamin D3 (orally, 17.8μg/kg) for eight weeks, fourth group (pregnant rats) administrated Vitamin D3 (orally, 8.9μg/k) for eight weeks, fifth group (pregnant rats) administrated Vitamin D3 (orally, 17.8μg/kg) for eight weeks, and then killed. The results showed high significant increased (P less than 0.05) in levels of lipid profile (total cholesterol, total glyceride, high density lipid (HDL), low density lipid (LDL) very low density lipid (VLDL)), especially in pregnant female rats (third and fifth groups) compared with control group. On the other hand, the results showed significant changes (P less than 0.05) in levels of malondialdehyde (MDA), Superoxide dismutase (SOD) and catalase especially in pregnant female rats (third and fifth groups) compared with control group. It was concluded that the prolong using and overdose of Vitamin D3 lead to elevated the lipid profile and oxidative stress in rats especially in pregnant female rats.