•Cefovecin administered to Patagonian sea lions shows a prolonged kinetic profile.•The half-life calculated for cefovecin varied from 11.3 to 21.6 days depending on the dose and route used.•Drug dosing should be linked to the susceptibility of the pathogen involved.•No adverse effects were observed following SC or IM cefovecin administration. Cefovecin is a third-generation cephalosporin developed as an aqueous solution for use by the subcutaneous route in dogs and cats. This study evaluated the duration of cefovecin plasma concentrations after single intramuscular (IM) or subcutaneous (SC) injection at different doses in 10 Patagonian sea lions (Otaria flavescens). Blood samples were collected serially from the day of the injection up to 60–90 days post-injection. Plasma drug concentrations were determined by high performance liquid chromatography–UV detection and pharmacokinetic parameters were calculated by non-compartmental analysis. No reactions or side effects associated with the drug were observed in any of the studied animals. Both routes showed very similar pharmacokinetic behaviour. Elimination half-life (11.3–21.6 days, SC; 13.1–15.9 days, IM) and mean residence time (17.6–36.8 days SC; 16.5–25.4 days IM) were, in all cases and doses, considerably longer than those previously reported for any other species. Based on these findings, and preliminary data on specific pathogen sensitivity, cefovecin was found to be a very promising antimicrobial for Patagonian sea lions, in particular those that are difficult to access or that are under certain rehabilitation conditions.