BACKGROUNDTicks, which are obligate blood‐feeding parasites, transmit a wide range of pathogens during their hematophagic process. Certain enzymes and macromolecules play a crucial role in inhibiting several tick physiological processes, including digestion and reproduction. In the present study, genes encoding type 2 cystatin were cloned from the tick Haemaphysalis doenitzi and characterized, and the potential role of cystatin in tick control was assessed.RESULTSTwo cystatin genes, HDcyst‐1 and HDcyst‐2, whose open reading frames are 390 and 426 bp, respectively, were successfully cloned from H. doenitzi. Among the midgut, salivary glands, Malpighian tubules, and ovaries of ticks, the relative expression of HDcyst‐1 and HDcyst‐2 was highest in the midgut and salivary glands, respectively. Lipopolysaccharide injection and low‐temperature stress elevated cystatin expression in ticks. Both recombinant cystatin protein rHDcyst‐1 and rHDcyst‐2 vaccines increased antibody levels and significantly reduced tick engorgement and egg mass weights in immunized rabbits infested with H. doenitzi. Particularly, rHDcyst‐1 significantly prolonged tick engorgement time by 1 day and reduced egg‐hatching rates by 16.9%. Collectively, rHDcyst‐1 and rHDcyst‐2 protein vaccinations provided 64.1 and 51.8% protection to adult female ticks, respectively.CONCLUSIONThis is the first report on the immunological characterization of cystatin proteins and gene sequencing in H. doenitzi. Cystatin proteins are promising antigens that have the potential for use as vaccines for H. doenitzi infestation control.This article is protected by copyright. All rights reserved.