The apicomplexan parasite Babesia bovis is responsible for bovine babesiosis, a poorly controlled tick-borne disease of global impact. The widely conserved gametocyte protein HAPLESS2/GCS1 (HAP2) is uniquely expressed on the surface of B. bovis sexual stage parasites and is a candidate for transmission-blocking vaccines (TBV). Here, we tested whether vaccination of calves with recombinant HAP2 (rHAP2) interferes with the transmission of B. bovis by competent ticks. Calves vaccinated with rHAP2 (n = 3), but not control animals (n = 3) developed antibodies specific to the vaccine antigen. Vaccinated and control animals were infested with Rhipicephalus microplus larvae and subsequently infected with virulent blood stage B. bovis parasites by needle inoculation, with all animals developing clinical signs of acute babesiosis. Engorged female ticks fed on the infected calves were collected for oviposition, hatching, and obtention of larvae. Transmission feeding was then conducted using pools of larvae derived from ticks fed on rHAP2-vaccinated or control calves. Recipient calves (n = 3) exposed to larvae derived from control animals, but none of the recipient calves (n = 3) challenged with larvae from ticks fed on rHAP2-vaccinated animals, developed signs of acute babesiosis within 11 days after tick infestation. Antibodies against B. bovis antigens and parasite DNA were found in all control recipient animals, but not in any of the calves exposed to larvae derived from HAP2-vaccinated animals, consistent with the absence of B. bovis infection via tick transmission. Overall, our results are consistent with the abrogation of parasite tick transmission in rHAP2-vaccinated calves, confirming this antigen as a prime TBV candidate against B. bovis.