The present study evaluated the effects of single-dose marbofloxacin in protecting horses against fever associated with transportation using 48 healthy Thoroughbreds. All horses were premedicated with interferon-α (0.5 U/kg, sublingually, every 24 hr) for 2 days before transportation and on the day of transportation. Horses were randomly assigned to receive marbofloxacin (2 mg/kg, IV, once; MRFX group), enrofloxacin (5 mg/kg, IV, once; ERFX group) or saline (0.9% NaCl) solution (10 ml, IV, once; control group) ≤1 hr before being transportation. Each group contained 16 horses (8 males, 8 females). Horses were transported 1,210 km using commercial vans over the course of approximately 26 hr. Clinical examinations and hematologic analyses were performed on all horses both before and after transportation. Post-transportation neutrophil to lymphocyte ratios were significantly lower in horses in the MRFX group compared with the control horses. The serum amyloid A levels were significantly lower in horses in the MRFX group and ERFX group compared with the control horses. Regarding the post-transportation rectal temperatures, fever was detected in 0 horses and 1 horse in the MRFX and ERFX groups, respectively, whereas fevers exceeding 39.1°C were detected in 2 horses in the control group. Additionally, the number of essential post-transportation treatments provided by veterinarians was reduced 3-fold in the MRFX and ERFX groups compared with the saline group. MRFX provided ERFX-like protection against fever associated with long-distance transportation, yielding significantly better protection than saline. Administration of MRFX just before transportation deserves a further study for efficacy in preventing horse fever associated with transportation.