Thrombostasin (TS) is a previously characterized anticlotting protein with multiple isoforms found in the saliva of horn flies. In this report, the effect of TS isoforms on blood feeding was assessed using individual flies that carried corresponding ts allelles. Laboratory studies of horn fly blood feeding were conducted using colony-reared flies fed on New Zealand White (NZW) rabbits. After timed 20-min feeding periods, each fly was characterized for gender, blood volume uptake, and ts genotype. The results showed that mean blood volumes obtained by individual flies were not related to fly gender but were correlated to the ts genotype(s) carried by each fly. A fly having one or both fe alleles coding for the TS9 isoform took less blood than those possessing one or both fe alleles coding for the TS10 isoform. These results confirm the significant role that TS plays in horn fly feeding and highlight the differential impact of TS protein isoforms that vary by as few as three amino acids.