Ivermectin (IVM) is an anthelmintic compound commonly used off-label in mules due to its broad-spectrum of activity. Despite the general use of IVM in mules with the same dose and route of administration licensed for horses, significant pharmacokinetic differences might exist between horses and mules, as already observed for donkeys. The aim of the present study was to evaluate the pharmacokinetic profile and anthelmintic efficacy of an oral paste of IVM in mules naturally infected with cyathostomins. Fifteen adult mules with fecal egg counts (FEC) ≥ 200 eggs per gram (EPG), with exclusive presence of cyathostomins, were included in the study. All mules were orally treated with IVM according to the manufacturer's recommended horse dosage (200 µg/kg body weight). FECs were performed before (day-10 and day-3) and after treatment at days 14 and 28 by using a modified McMaster method. The FEC reduction (FECR%) was also calculated. Blood samples were collected from five animals at various times between 0.5 h up to 30 days post treatment to determine pharmacokinetic parameters. The maximum IVM serum concentration (Cmax) was 42.31 ± 10.20 ng/mL and was achieved at 16.80 ± 9.96 h post-treatment (Tmax), area under the curve (AUC) was 135.56 ± 43.71 ng × day/mL. FECR% remained high (>95%) until the 28th day.