The aim of this trial was to study the concentration of zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), selenium (Se), cobalt (Co) and iodine (I) in milk and blood serum of lactating donkeys, taking into account the effects of lactation stage and dietary supplementation with trace elements. During a 3-month period, 16 clinically healthy lactating donkeys (Martina-Franca-derived population), randomly divided into two homogeneous groups (control (CTL) and trace elements (TE)), were used to provide milk and blood samples at 2-week intervals. Donkeys in both groups had continuous access to meadow hay and were fed 2.5 kg of mixed feed daily, divided into two meals. The mixed feed for the TE group had the same ingredients as the CTL, but was supplemented with a commercial premix providing 163 mg Zn, 185 mg Fe, 36 mg Cu, 216 mg Mn, 0.67 mg Se, 2.78 mg Co and 3.20 mg I/kg mixed feed. The concentrations of Zn, Fe, Cu, Mn, Se, Co and I were measured in feeds, milk and blood serum by inductively coupled plasma-MS. Data were processed by ANOVA for repeated measures. The milk concentrations of all the investigated elements were not significantly affected by the dietary supplementation with TE. Serum concentrations of Zn, Fe, Cu Mn and Se were not affected by dietary treatment, but TE-supplemented donkeys showed significantly higher concentrations of serum Co (1.34 v. 0.69 μg/l) and I (24.42 v. 21.43 μg/l) than unsupplemented donkeys. The effect of lactation stage was significant for all the investigated elements in milk and blood serum, except for serum manganese. A clear negative trend during lactation was observed for milk Cu and Se concentrations (−38%), whereas that of Mn tended to increase. The serum Cu concentration was generally constant and that of Co tended to increase. If compared with data reported in the literature for human milk, donkey milk showed similarities for Zn, Mn, Co and I. Furthermore, this study indicated that, in the current experimental conditions, the mineral profile of donkey milk was not dependent on dietary TE supply.