In this study, the pharmacokinetic disposition and bioavailability of marbofloxacin (MB) were determined in lambs after single intravenous (IV), intramuscular (IM), and subcutaneous (SC) administrations at a dose of 3 mg/kg. The plasma concentration of MB was measured using high-performance liquid chromatography-UV, and the pharmacokinetic parameters were analyzed using a non-compartmental analysis. Following IV, IM, and SC administrations, the mean terminal half-life (t1/2ʎz) was 11.48, 12.64, and 24.86 h, respectively, and the mean residence time (MRT) was 7.27, 7.81, and 10.11 h, respectively. The bioavailability (F) was 96.01 and 126.39%, after IM and SC administration, respectively. This study showed that SC administration of MB at a dose of 3 mg/kg exhibited flip-flop pharmacokinetics in lambs. These results suggested that MB could be useful in the treatment of severe systemic infections, such as those with M. haemolytica (MIC = 0.035 μg/mL), in lambs since high AUC0-24/MIC and Cmax/MIC ratios were achieved after IV and IM administration at 3 mg/kg. However, MB administration (3 mg/kg) via the IV, IM, and SC routes might not be effective in the treatment of respiratory infections caused by organisms with MIC90 value in lambs.