The objective of this study was to evaluate kinetics and the residual decline of tylosin in milk and plasma of lactating Najdi ewes following single intramuscular injection of tylosin at the dose of 10 mg/kg. Blood and milk samples were collected from the ewes before and at different time intervals after treatment. Tylosin concentrations were determined by microbiological agar plate assay using Bacillus subtilis ATCC 6633 as the test organism. The pharmacokinetic parameters were processed using the methods of least square and statistical moments. The plasma levels of tylosin against time were adequately described by a one compartment open model. The mean obtained values indicated a fairly low area under the plasma concentration-time curve (AUC) (3.0 μg.h/ml) and the maximum plasma concentration (Cmax) of 0.63 μg/ml with Tmax of 1.33 h. The plasma elimination half-life (t1/2el) and the mean residence time (MRT) were 2.3 h and 3.9 h, respectively. A different pattern was shown for milk, in which measurable residual levels are found in all animals up to 72 h after treatment. The mean value of milk AUC was 88.1 μg.h/ml and the t 1/2el was 3.3 h. In vitro mean plasma and milk proteins binding of tylosin were 19.3 and 30.2%, respectively. The milk withdrawal period of tylosin in lactating Najdi ewes should be at least 72 h to avoid risks in consumers.