Recombinant Glutathione S-transferase of Haemaphysalis longicornis (rGST-Hl) was expressed in Escherichia coli, purified by affinity chromatography and used in the immunization of cattle. Western blot analysis showed positive antibody response in cattle immunized with rGST-Hl. The tests also showed that immunized bovine sera recognize native Rhipicephalus microplus proteins in different tissue extracts. Furthermore, the vaccine potential of rGST-Hl was investigated against infestation of Hereford cattle by R. microplus. Vaccination of cattle with rGST-Hl conferred partial cross-protection immunity against R. microplus. Considering the effect on number of engorged ticks, egg laying capacity and egg fertility, the overall efficacy of vaccination was of 57%, as compared with control group.