Mid gut antigens of Hyalomma marginatum isaaci were prepared and purified by gel filtration chromatography using Sephadex G-200. A total of 70 fractions were eluted, out of which only eleven (F7-F16 and F18) were observed to be immunogenic by ELISA. The SDS-PAGE profile of mid gut antigen revealed 3 bands at 198, 97 and 20 kDa. Western blot analysis of midgut antigen of H. m. isaaci using the hyperimmunized rabbit serum recognized 2 immunoreactive proteins with molecular weights of 198 and 97 kDa. Adjuvant saponin induced better cell mediated and humoral immune response than Montanide ISA 50 adjuvant. In Saponin group, a significant leucocyte migration inhibition was observed from 21 DPI with peak inhibition on 42 DPI and maintained at moderate levels till 91 DPI, while a significant inhibition was noted on 28 DPI with the peak value on 35 DPI, which declined on 56 DPI in Montanide group. Calves immunized with midgut antigen of H. m. isaaci failed to confer protection against larval and nymphal stages of B. microplus but evoked moderate protection in terms of morality (29.50 ± 2.12 % in Saponin group and 24.00 ± 5.65 % in Montanide group) against B. microplus ticks.