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This study was conducted to investigate the effects of feeding system and NDF levels of TMR on blood metabolites in Holstein heifers. Fifty heifers were assigned to one of five treatments according to feed type and level of NDF: TMR (total mixed ration), CON (concentration + mixed forage), T1 (≥ 53% NDF), T2 (50∼53% NDF), and T3 (≤ 50% NDF). Although GLU concentrate was not effected by feed type, increased significantly as the level of NDF decreased. There was no effect on concentrate of blood metabolite related with protein. As the level of NDF decreased, the blood ALB concentration was increased (p<0.05). The concentration of blood CHOL was higher in CON than those of TMR (p<0.05), and increased as the level of NDF decreased. The concentration of blood lipid was higher in CON than those of TMR and increased at lower level of NDF. The concentration of blood ALT were significantly lower in T3 than T1 and T2 (p<0.05). Thus, the results of this study suggest that the feeding system and NDF level may affect the blood metabolite concentration; however, the feed intake and other nutrient levels should also be considered.