We have previously undertaken preliminary characterization of two Rhipicephalus appendiculutus serine protease inhibitors (RAS-1 and -2) as anti-tick vaccine candidates. In this study, to clarify this hypothesis, we generated and further characterized recombinant RAS-1 and -2 (rRAS-1 and -2) and tested their potency as a cocktail anti-tick vaccine in cattle. RT-PCR analysis showed that RAS-1 and -2 mRNA transcripts are expressed during all life cycle stages of ticks, independent of sex. As judged by SDS-PAGE rRAS-1 and -2 migrated as a molecular weight of around 64 and 60 kDa protein, respectively, considering that the expression vector produced a recombinant protein fused with 18-22 kDa TRX protein. RAS-1 and -2 were found not to be secreted into the bite site as determined by the reactivity of anti-tick saliva sera to rRAS-1 and -2, suggesting that both proteins are concealed antigens. Vaccination of cattle with a combination of rRAS-1 and -2 conferred significant protective immunity against ticks, resulting in 61.4% reduction in nymph engorgement rate, and in 28 and 43% increased mortality rate in adult female and male ticks, respectively. This is the first report on an anti-tick vaccine trial using a combination of two different serpins derived from R. appendiculatus, and using cattle as a natural host.