Osteoporosis features include reduction of bone mass and increased predisposition to fractures. Osteoporosis is considered as one of frequent diseases developing in menopausal women and old men. The values of peak bone mass are the determinant of osteoporosis development. It is estimated that the peak bone mass is reached at the age between 16 and 30 years. (NIH Consensus, 2001). Insufficient intake of calcium and vitamin D in diet, with reduced sun exposure, low physical activity, low body weight are well documented risk factors for this disease in human. For that reason, in osteoporosis prevention it should be tried to ensure adequate supply of calcium in diet from early life, with adequate supply of vitamin D, as factors determining adequate mineralization of osseous tissue. The study was carried out for assessing changes of the levels of calcium and 25-OH cholecalciferol in serum replacing butter in usual diet with margarine enriched with vitamin D. After a period of diet stabilization the study group received first diet with butter, followed by diet with margarine in place of butter. Throughout the whole experiment the diet contained unchanged amounts of dairy products as a source of alimentary calcium. In the experiment 41 young healthy men aged 22.6 +/- 1.2 years participated. Every subject consumed in a period of 4 weeks 30 g of butter daily, and during the following 4 weeks margarine 30 g daily. Substitution of margarine for butter in usual diet raised 25-OH cholecalciferol level in serum by 5.56 ng/ml, that is 32.4% (p < 0.001), with consequent 0.12 mg (5%) rise of serum calcium level (p < 0.05). The intake of calcium with diet from dairy products was unchanged throughout the experiment 576-581 mg daily, that is 72% of the recommended intake. The obtained results suggest that independently of the amount of vitamin D intake with food and calcium with diet substitution of margarine enriched with vitamin D for butter was beneficial raising serum vitamin D level, and at the same time increasing calcium absorption from food. It can be accepted that the obtained results could support the opinion on beneficial effects of the consumption of high-quality margarine in daily diet as prevention of the development not only of atherosclerosis but also osteoporosis.