In Italy, the interest for jennet milk production has recently developed. An 18-month-long experiment was carried out on a jennet farm near Milo (CT), where 24 jennets, which derived from the Ragusana breed, were tested for milk yield and composition over an entire lactation period. The jennets were fed with hay and concentrate in a large paddock. From the 28th post-foaling day to the end of the lactation, the jennets were machine-milked twice a day with an in-between milking interval of 5 h. The milk amount from each jennet was recorded every 3 weeks and individual samples were collected and analyzed for fat, protein, casein, non-proteic nitrogen, lactose and somatic cell count. This study showed that jennets at Sicilian latitudes are not seasonal polyestrous. The daily milk yield, the length of lactation and the milk characteristics varied depending on the foaling season. The total average milk production was 490 ± 36 kg in 295 ± 12 post-foaling days, considering two milking records per day. During the lactation, milk yield decreased constantly from 1.98 to 1.28 kg/jennet per day. When looking at the jennet milk quality during lactation, the percentage of fat and protein decreased, while the lactose percentage increased, according to a tendency apparently unique for equines when compared to the ruminants. When looking at the productive season, spring generally gave the best qualitative and quantitative results. Based on these results, jennet milk yield and quality could be improved; furthermore, jennet milk production may turn out to be a profitable business.