The study evaluated the effect of graded levels of vitamin E supplementation on the serum α-tocopherol, retinol concentrations and immune response of buffalo calves. In a completely randomized design, 15 male Murrah buffalo calves (14-15 months old; 194.0 ± 3.5 kg average body weight) were divided into 3 equal groups on the basis of their body weights. All the calves were fed on wheat straw and concentrate mixture to meet their nutrients requirements. In addition, all the calves in group II and III were supplemented with 300 and 600 IU of feed grade DL- α-tocopheryl acetate/d, respectively. Blood samples were collected at zero day and subsequently at 30 days intervals up to 120 days to estimate serum α-tocopherol and retinol concentration. To assess humoral immune response, all the buffalo calves were sensitized with formalin inactivated Pasteurella multocida antigen and blood was collected on 0, 7, 14, 21 and 28 days post vaccination to study antibody production using indirect ELISA, whereas cell mediated immune response of buffalo calves was assessed by in vivo delayed type hypersensitivity (DTH) reaction against phytohaemaglutinin-P (PHA-P). Retinol concentration (μg/ml) in blood serum of three groups of calves was statistically similar (P>0.05), whereas the cumulative group mean serum α-tocopherol concentration (μg/dl) was significantly (P<0.01) higher in vitamin E supplemented groups as compared to control group. The pooled mean ELISA antibody titer against Pasteurella multocida was also significantly (P<0.05) higher in buffalo calves given vitamin E as compared to control group calves, but cell mediated immune response was similar ((P>0.05) in three groups. Results of the present study indicated that vitamin E supplementation increased the α-tocopherol concentration in serum and also enhanced the humoral immune response of buffalo calves.