Enzyme-induced hemolysis has been shown to occur in the midgut of ticks; however, little is known about the molecular basis for hemolytic activity. We report here the molecular and reverse genetic characterization of a hemolytic midgut serine proteinase, HlSP, recently identified from the ixodid tick Haemaphysalis longicornis. Endogenous HlSP was found in the midgut lumen and its contents, indicating that HlSP is extracellularly secreted. Recombinant H. longicornis serine proteinase (rHlSP) expressed in Escherichia coli showed dose-dependent hemolytic activity towards rabbit erythrocytes, with a maximum hemolysis of 94.5% within 1 h in vitro. Tests of pH dependency showed that rHlSP displayed optimal activity at pH 6.0. In binding assays, rHlSP showed high affinity to band 3, which shares the major erythrocyte membrane proteins. Disruption of HlSP-specific mRNA by RNA interference resulted in inhibition of the degradation of host erythrocyte membranes by endogenous HlSP in the knock-down ticks, indicating that HlSP plays a crucial role in the hemolysis in the midgut of haematophagous ticks. Our results suggest that HlSP may be essential for initiating the proteolytic cascade for the degradation of the host blood-meal.