The traditional utilization of donkeys (Equus asinus) as dairy animals has recently attracted substantial scientific interest with regard to human nutrition. Donkey milk is well tolerated by infants with cows’ milk allergy, useful in the treatment of human immune-related diseases, in the prevention of atherosclerosis, and in-vitro studies showed an anti-proliferative effect. Active 3-3′-5-triiodothyronine (T3) in colostrum and milk could play different physiological roles, systemic and paracrine, for both the mother and the suckling offspring. The aim was to evaluate whether thyroid hormones (TH) concentrations in milk and blood of lactating donkeys change with the advancing lactation and whether they can be affected by dietary supplementation with several trace elements, some of them directly involved with TH synthesis (I), metabolism (Se) and action (Zn). Sixteen lactating jennies were divided into two groups (CTL and TE). Mixed feed for TE was added with Zn, Fe, Cu, Mn, I, Se and Co. Every 2 weeks milk and blood samples were collected at 11·00. Total concentrations of T3 in milk (T3M) and T3 and T4 in plasma (T3P and T4P) were assayed using ELISA kits, validated for the donkey species. T3M was not correlated with TH concentrations in blood, did not change with the stage of lactation, and was significantly higher in TE (4·09±0·07 ng/ml, mean±se) than in CTL group (3·89±0·08 ng/ml). T4P (81·8±5·2 ng/ml) and T3P (15·2±1 ng/ml) significantly changed with time, but were not significantly affected by dietary treatment. T3P/T4P ratio was significantly lower in TE group. This study indicates that in donkey milk the concentration of T3, a human-like bioactive compound, can be affected by trace elements intake.