A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of pathenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to a Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delays bovine plasma clotting time and inhibits both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene expresses at all stages of the tick except for the egg stage and mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene led to a 2-day extension of the tick blood feeding period, and 27.7% of the ticks did not successfully complete the blood feeding. These findings indicate that the newly indentified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.