Florfenicol was administered to five heifers intramuscularly at a dose rate of 20 mg/kg bwt and following wash‐out period, subcutaneously at a dose rate of 40 mg/kg bwt. Blood plasma samples were collected from heifers before injection of florfenicol and up to 120 h after intramuscular (IM) injection and up to 264 h after subcutaneous (SC) injection. Florfenicol concentrations in plasma were measured by high‐performance liquid chromatography with mass‐spectrometric detection. Pharmacokinetics of florfenicol was estimated using non‐compartment analysis. Mean maximum plasma concentration, area under the concentration–time curve and elimination half‐life for florfenicol were 3.2 μg/ml, 101.5 μg × h/ml and 24.5 h, respectively, after IM injection at 20 mg/kg bwt, and 2.7 μg/ml, 194.5 μg × h/ml and 103.8 h, respectively, after SC injection at 40 mg/kg bwt. The obtained results indicated that both administration routes provided comparable bioavailability, whereas SC route was attributed with lower peak levels and markedly slower absorption of florfenicol from injection site. Both administration routes provided plasma florfenicol levels which are expected to be effective against prevalent infectious agents of cattle.