A new Kunitz‐type serine protease inhibitor, Rhipilin‐2, was identified in the tick Rhipicephalus hemaphysaloides. The cDNA sequence of Rhipilin‐2 is 693 bp, and it encodes a deduced 195 amino acid protein with a size of 22 kDa. Bioinformatic analysis shows that Rhipilin‐2 belongs to the Kunitz‐type family of inhibitors, containing one Kunitz domain with homology to the tissue factor pathway inhibitor. Using Real time polymerase chain reaction (Real time‐PCR), Rhipilin‐2 mRNA transcripts were detected in tick salivary glands and midgut. Blood feeding induced transcript expression. The recombinant protein was expressed in insect Sf9 cells and confirmed by immunofluorescence test and Western blot analysis with an anti‐His antibody. The purified recombinant Rhipilin‐2 inhibited serine protease trypsin and elastase, but not thrombin. The anticoagulant activity of Rhipilin‐2 was shown by delaying normal clotting of rabbit plasma in the activated partial thromboplastin time tests. These results indicate that Rhipilin‐2 is a novel Kunitz‐type serine protease inhibitor involved in tick blood feeding.