The present study investigates the changes in blood cholesterol and total lipids concentrations, as well as changes in milk fat, protein, lactose and total solids content of sows after the long-term administration of virginiamycin (VM) in the feed. Seventy-two (72) healthy Dalland gilts, in 18 groups of four (2 + 2) siblings were used in total. The gilts of each group of siblings were randomly allocated to one of the following four dietary treatments: VM0 = negative control, virginiamycin 0 mg/kg, VM20 = virginiamycin 20 mg/kg feed, VM40 = virginiamycin 40 mg/kg feed and VM60 = virginiamycin 60 mg/kg of feed. Treatments started at the age of 6 months covering three complete breeding cycles (up to the third weaning). The first eight groups of siblings (eight gilts per treatment/32 gilts in total) were used for blood sampling (blood group), while the remaining 10 groups of siblings (10 gilts per treatment/40 gilts in total) were used for milk sampling (milk group). Feeds given at insemination, pregnancy and lactation did not contain any other antibacterial or performance enhancer. The results indicate that all three levels of VM supplementation of gilt/sow feed influenced certain blood parameters by increasing (P < 0.05) both cholesterol and total lipids concentrations on the 30th and 60th day of each pregnancy, at each farrowing and at each weaning. The effect of dietary VM on these blood parameters remained constant throughout three consecutive parities. Moreover, the results indicate a beneficial effect of dietary VM on the milk quality of the sows during the third lactation by increasing (P < 0.05) (i) the mean fat content, (ii) the mean protein content, (iii) the mean lactose content and (iv) the mean total solids content. The highest (P < 0.05) levels in both blood and milk parameters were noticed at the inclusion level of 40 mg/kg of feed.