Objectives were to determine the effect of supplementing 2 sources of vitamin D, cholecalciferol (CH) or calcidiol (CA), at 1 (1mg) or 3 mg/d (3mg) prepartum on concentrations of vitamin D metabolites in plasma, measures of innate immune function, and leukocyte mRNA expression. Parous Holstein cows (n = 99) were assigned to a daily treatment administered as top-dress containing either 1 or 3 mg of CH (CH1 or CH3) or of CA (CA1 or CA3) from 250 d of gestation until calving. Plasma concentrations of vitamin D, immune cell population in blood, cell adhesion markers, and granulocyte phagocytosis and oxidative burst were evaluated pre- and postpartum. The mRNA expression in leukocytes was determined at 270 d of gestation and 3 d postpartum for genes involved in cell migration, pathogen recognition receptors, cell signaling, cytokines, antimicrobial mechanisms, oxidative burst, and Ca and vitamin D metabolism. Concentrations of vitamin D3 increased in cows fed CH, whereas those of 25-hydroxyvitamin D3 increased in cows fed CA. Percentage of granulocytes from total leukocytes differed with amount of vitamin D pre- (1mg = 24.5 vs. 3mg = 37.9%) and postpartum (1mg = 22.0 vs. 3mg = 31.0%), thus shifting mononuclear cells in the opposite direction pre- (1mg = 75.5 vs. 3mg = 62.1%) and postpartum (1mg = 78.0 vs. 3mg = 69.0%). Granulocytes displaying phagocytosis (1mg = 69.0 vs. 3mg = 62.9%) and intensity of phagocytosis prepartum (1mg = 7.46 vs. 3mg = 7.28) tended to be less in cows fed 3mg compared with 1mg. During prepartum, CA increased mRNA expression of genes related to cell adhesion and migration (CD44, ICAM1, ITGAL, ITGB1, LGALS8, SELL), pathogen recognition receptor (NOD2, TLR2, TLR6), cell signaling (FOS, JUN, NFKB2), cytokine signaling (IL1B, IL1R1, IL1RN), antimicrobial mechanisms (CTSB, LYZ), and Ca metabolism (ATP2B1, STIM1, TRPV5) compared with CH. Similarly, postpartum, CA increased mRNA expression of genes related to cell adhesion and migration (CXCR2, SELL, TLN1), cell signaling (AKT2), cytokines (CCL2, IL1R1, ILRN), antimicrobial mechanisms (DEFB3), oxidative burst (RAC2), and calcium metabolism (CALM3) compared with CH. Feeding additional vitamin D in the last 3 wk of gestation changed the profile of blood leukocytes and attenuated granulocyte phagocytosis during the transition period, whereas supplementing CA prepartum increased mRNA expression of genes involved in immune cell function, including genes related to pathogen recognition and antimicrobial effects of leukocytes.