Objectives were to test the effects of feeding vitamin D₃ compared with 25-hydroxyvitamin D₃ on physiological and immune responses of dairy calves to an intravenous endotoxin challenge. Forty-five Holstein bull calves (<7 d of age) were blocked into weekly cohorts and randomly assigned to one of 5 daily supplements; control (CON, 0.25 mg of vitamin D₃/kg BW), and 2 levels (1.5 or 3.0 μg/kg BW) of vitamin D3 (VitD), or 25-hydroxyvitamin D₃ (25D) added to the CON treatment resulting in the following treatment groups: CON, Vit1.5, VitD3.0, 25D1.5, 25D3.0. Calves were fed milk replacer until weaning at 56 d of age and had ad libitum access to water and a starter grain throughout the experiment. Treatments were added to milk replacer once daily until weaning, then to starter grain thereafter. On d 91 of the experiment, calves were challenged with lipopolysaccharide (0.1 μg/kg BW) via intravenous injection and physical and plasma measures were evaluated in response to the challenge. Data were analyzed with mixed model that included fixed effects treatment and time and random effects of calf within block. Average rectal temperatures from 1 to 72 h after challenge were 39.1, 39.1, 38.8, 38.9 and 39.1 ± 0.1°C, for CON Vit1.5, Vit3.0, 25D1.5 and 25D3.0 calves, respectively. A similar pattern was observed for reactive oxygen metabolites in serum and the interaction between source and dose was significant (P < 0.01) for rectal temperatures and reactive oxygen metabolites in blood such that responses were greater in VitD1.5 and 25D3.0 calves compared with VitD3.0 and 25D1.5 calves. Concentrations of Ca, NEFA, glucose and haptoglobin in serum changed over time but were not affected by treatment; however, serum P was decreased (P < 0.01) in CON calves (2.3 ± 0.1 mM) compared with VitD and 25D treated calves (2.6, 2.5, 2.4 and 2.5 ± 0.1 mM). Leukocyte counts and expression CD11b, CD14, CD62L proteins did not differ between treatments. Feeding VitD at 3.0 μg/kg BW and 25D at 1.5 μg/kg BW resulted in better tolerance of calves to an endotoxin challenge compared with 1.5 μg/kg BW VitD or 3.0 μg/kg BW 25D.