Background: Potomac horse fever (PHF) is a potentially fatal enterocolitis of horses caused by Neorickettsia risticii. The disease was originally recognised almost 40 years ago in the state of Maryland in the US. It is now known to occur in many areas of North America, as well as having been described in South America and Europe. Monocomponent PHF vaccines are available, but clinical protection with vaccination has been reported to be inconsistent. Objectives: This study was designed to assess the immunogenicity of a commercially available Potomac Horse Fever (PHF) vaccine when administered as either a monovalent PHF vaccine simultaneously co-administered with a separate monovalent Rabies vaccine or as a multivalent PHF/Rabies vaccine in horses. Study design: Randomised parallel group trial. Methods: Ninety-one client or University owned horses participated in this open-label randomised study, with 45 horses receiving the monovalent vaccines at separate sites and 46 receiving the multivalent vaccine at a single site. Serum PHF IFA titres were determined twice prior to vaccination and at 1, 2 and 3 months after vaccination. Results: Both vaccination protocols exhibited poor immunogenicity, with only one-third of all the animals demonstrating seroconversion, defined as an increase in titre of greater than 400 over baseline, at any time point after vaccination. The monovalent PHF vaccine exhibited significantly greater immunogenicity in terms of the number of horses exhibiting seroconversion, as compared to the multivalent vaccine, at one (20 vs. 11, P = 0.03) and two (18 vs. 9, p = 0.02) months post vaccination. The monovalent PHF vaccine also exhibited significantly greater immunogenicity in terms of the median (interquartile range) IFA titres, as compared to the multivalent vaccine, at one (800 [200–1600] vs. 400 [200–800], P = 0.009) and 2 months (400 [200–1600] vs. 400 [100–800], P = 0.02) post vaccination. There was no significant difference between groups at 3 months in either seroconversion rate or median IFA titers. Main limitations: This study did not assess the actual protective effects of PHF vaccination but rather used the serologic response to vaccination as a surrogate biomarker of immunity. Conclusions: The multivalent PHF/Rabies vaccine exhibited lower immunogenicity as compared to the monovalent PHF vaccine co-administered with a separate Rabies vaccine.