The objective of this study was to determine the pharmacokinetics of a long-acting formulation of ceftiofur crystalline-free acid (CCFA) following intramuscular injection in ball pythons (Python regius). Six adult ball pythons received an injection of CCFA (15 mg/kg) in the epaxial muscles. Blood samples were collected by cardiocentesis immediately prior to and at 0.5, 1, 2, 4, 8, 12, 18, 24, 48, 72, 96, 144, 192, 240, 288, 384, 480, 576, 720, and 864 hr after CCFA administration. Plasma ceftiofur concentrations were determined by high-performance liquid chromatography. A noncompartmental pharmacokinetic analysis was applied to the data. Maximum plasma concentration (C_max) was 7.096 ± 1.95 μg/ml and occurred at (T_max) 2.17 ± 0.98 hr. The area under the curve (0 to infinity) for ceftiofur was 74.59 ± 13.05 μg*h/ml and the elimination half-life associated with the terminal slope of the concentration-time curve was 64.31 ± 14.2 hr. Mean residence time (0 to infinity) was 46.85 ± 13.53 hr. CCFA at 15 mg/kg was well tolerated in all the pythons. Minimum inhibitory concentration (MIC) data for bacterial isolates from snakes are not well established. For MIC values of ≤0.1 μg/ml, a single dose of CCFA (15 mg/kg) provides adequate plasma concentrations for at least 5 days in the ball python. For MICs ≥0.5 μg/ml, more frequent dosing or a higher dosage may be required.