Background: Moxifloxacin is a new fourth-generation 8-methoxy fluoroquinolone developed primarily for the treatment of community-acquired pneumonia and upper respiratory tract infections. The aim of the study was to investigate the plasma pharmacokinetics characteristic of moxifloxacin in calves, after intravenous, intramuscular and subcutaneous administration of a single dose. Meanwhile, plasma protein binding and bioavailability of moxifloxacin were also estimated. Methods: Plasma concentrations of moxifloxacin were measured using a modified HPLC method, and the extent of plasma protein binding was determined in vitro using ultrafiltration. Results: Following intravenous administration, the half life of elimination, the volume of distribution at steady state and the area under the curve were 3.29 h, 0.94 l/kg and 24.72 μg·h/ml, respectively. After intramuscular and subcutaneous administration of moxifloxacin at the same dose, the peak plasma concentrations were 2.41 and 2.20 μg/ml and were obtained at 1.54 and 1.59 h, respectively. The systemic bioavailabilities were 87.19 and 75.94%, respectively. The in vitro plasma protein binding of moxifloxacin in plasma of calves was 27%. Conclusion: A high peak plasma concentration, area under the curve, rapid absorption and bioavailability following intramuscular and subcutaneous administration characterize the pharmacokinetics of moxifloxacin in calves.