Moderate exposure to sunlight increases vitamin-D status in the body and vitamin-D helps in absorption of calcium from intestine. This study was aimed to investigate the efficacy of vitamin-D and sunlight on blood parameters and serum biochemistry in mice. For this purpose, 50 adult Swiss albino mice were collected and randomly divided into five equal groups (Group A, B, C, D and E). Group A was considered as control and fed only on balanced normal feed. Group B was supplied with 15% butter in feed as a semi-synthetic source of vitamin-D, group C was exposed to direct sunlight (1.5 hours daily) and group D and E were supplied with oral synthetic form of vitamin-D @ 50 IU and 100 IU daily, respectively. After 120 days, blood and serum samples were collected for analysis. Our results showed that total erythrocytes count (TEC) and total leukocytes count (TLC) were found higher in group D but insignificant. Lymphocytes were significantly (P__ampersandsignlt;0.001) higher in group D but neutrophils was significantly (P__ampersandsignlt;0.01) higher in group B. The aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (AP) levels were also raised very sharply with increased level of vitamin-D in blood. AST and AP level were found significantly (P__ampersandsignlt;0.001) higher in group B but ALT level was significantly (P__ampersandsignlt;0.001) higher in group E. The lipid profile such as total cholesterol (TC), high density lipoprotein (HDL) and triglyceride (TG) were also analyzed. TC level was slightly higher in group C but significantly (P__ampersandsignlt;0.01) lower in group E HDL was significantly increases with increased level of vitamin-D in treatment groups (P__ampersandsignlt;0.01). TG level was somewhat higher in group D but not significant. From our results it can be concluded that sunlight and butter are the potential sources of vitamin-D. Supplementation of vitamin-D at certain level improves health status but excessive supplementation cause harmful effects on the body systems.