BACKGROUND/OBJECTIVES: The incidence of cardiovascular diseases (CVDs) has increased worldwide. Although a low serum vitamin D level is known to be associated with the risk of CVD, the mechanism is not well understood yet. The aim of this study was to determine the relationship of serum 25-hydroxyvitamin D(3) (25[OH]D) with homocysteine and apolipoprotein B (ApoB). SUBJECTS/METHODS: Of 777 subjects recruited from one health promotion center for routine heath exam from January 2010 to December 2016, 518 subjects were included in this study. Serum 25(OH)D, serum homocysteine, and other metabolic parameters including ApoB were analyzed. Simple and partial correlations were carried out after adjustments. Simple linear regression analysis was used for precise correlation of parameters. Multivariate regression analysis was done to know which factor (serum homocysteine or ApoB) was more related to serum 25(OH)D after adjustments. Finally, logarithms of homocysteine concentrations according to tertiles of serum 25(OH)D were compared. RESULTS: After sex and age adjustments, serum 25(OH)D showed negative correlations with serum homocysteine (r' = -0.114) and ApoB (r' = -0.098). In simple linear regression analysis, serum 25(OH)D showed a significant negative correlation with ApoB (P = 0.035). However, in multivariate regression analysis, serum 25(OH)D was significantly associated with serum homocysteine after adjustments (P = 0.022). In addition, serum homocysteine concentration was significantly high in the lowest 25(OH)D group (P = 0.046). CONCLUSION: Serum 25(OH)D concentration showed a stronger negative association with serum homocysteine than with ApoB.