The horn fly, Haematobia irritans (L.), is a blood-sucking livestock ectoparasite responsible for substantial livestock losses. In the present work, the potential use of recombinant hematobin (HTB), a horn fly salivary protein, as an antigen for cattle vaccination was investigated. In this trial, horn fly loads and H. irritans's blood intake were assessed in vaccinated (n = 4) and control (n = 4) crossbred dark-coated steers, which were naturally infected. The vaccinated group received a 1 ml subcutaneous injection of 100 µg of HTB protein emulsified in 500 µl of Incomplete Freund Adjuvant (AIF) on days 0 and 30. The control group received on the same days 1 ml of distilled water emulsified in 500 µl of AIF. The vaccinated group had significantly more HTB-specific IgG antibodies after the HTB booster and had a lower fly load than the control group (206 ± 23 vs. 285 ± 23 flies per animal, respectively). Blood intake by H. irritans did not differ between groups. In summary, these results suggest that vaccinating cattle with HTB could reduce cattle H. irritans load.