Vector ticks possess a unique system that enables them to digest large amounts of host blood and to transmit various animal and human pathogens, suggesting the existence of evolutionally acquired proteolytic mechanisms. We report here the molecular and reverse genetic characterization of a multifunctional cysteine protease, longipain, from the babesial parasite vector tick Haemaphysalis longicornis. Longipain shares structural similarity with papain-family cysteine proteases obtained from invertebrates and vertebrates. Endogenous longipain was mainly expressed in the midgut epithelium and was specifically localized at lysosomal vacuoles and possibly released into the lumen. Its expression was up-regulated by host blood feeding. Enzymatic functional assays using in vitro and in vivo substrates revealed that longipain hydrolysis occurs over a broad range of pH and temperature. Haemoparasiticidal assays showed that longipain dose-dependently killed tick-borne Babesia parasites, and its babesiacidal effect occurred via specific adherence to the parasite membranes. Disruption of endogenous longipain by RNA interference revealed that longipain is involved in the digestion of the host blood meal. In addition, the knockdown ticks contained an increased number of parasites, suggesting that longipain exerts a killing effect against the midgut-stage Babesia parasites in ticks. Our results suggest that longipain is essential for tick survival, and may have a role in controlling the transmission of tick-transmittable Babesia parasites. Ticks are important ectoparasites among the blood-feeding arthropods and serve as vectors of many deadly diseases of humans and animals. Of tick-transmitted pathogens, Babesia, an intracellular haemoprotozoan parasite causing a malaria-like disease, called babesiosis, gain increasing interest due to its zoonotic significance. When vector ticks acquire the protozoa via blood-meals, they invade midgut and undergo several developmental stages prior to exit through salivary glands. It has long been conceived that midguts of these ticks evolve diverse innate immune mechanisms and perform blood digestion critical for tick survival. A cysteine proteinase, longipain, was identified from the three-host tick Haemaphysalis longicornis, which shows potent parasiticidal activity. Longipain is localized in midgut epithelium and its expression is induced by blood feeding. This protein is passively secreted into midgut lumen where it exerts enzymatic degradation of blood-meals. A series of experiments unveil that longipain-knockdown ticks when fed on Babesia-infected dog, exhibited a significantly increased numbers of parasites compared with controls. Longipain has shown to interact on the surface of Babesia parasites in vitro and in vivo, and is thought to mediate direct killing of the parasites, suggesting that longipain may be a potential chemotherapeutic target against babesiosis and ticks themselves.