The present work was undertaken to evaluate the effects of Lactobacillus acidophilus supplementation of a milk substitute on the features of lamb rennet paste used for cheese making. Lipolysis in cheese manufactured with rennet paste from lambs receiving supplemented milk was also evaluated. Lambs were subjected to 3 different feeding regimens (mother suckling, MS; artificial rearing, AR; and artificial rearing with 7 log10 cfu/mL of Lb. acidophilus supplementation of the milk substitute, ARLb) and slaughtered at 20 and 40 d of age for each feeding treatment. Abomasa of the lambs were processed to rennet paste. Microbial loads, enzymatic activities (chymosin, pepsin, and lipases), and renneting characteristics of the lamb rennet paste were determined. Free fatty acids and conjugated linoleic acids were detected in cheese at 60 d of ripening. Addition of 7 log10 cfu/mL of Lb. acidophilus to the milk substitute was carried out successfully. Total recovery of viable cells was recorded in milk supplied daily to the lambs in the ARLb group. The ARLb rennet had greater amounts of lactobacilli than did the MS or AR rennet, irrespective of the slaughter age of the lambs, and the ARLb rennet had higher concentrations of lactococci when lambs were slaughtered at 40 d of age. Chymosin and lipase activities were also higher in ARLb rennet than in MS or AR rennet from lambs slaughtered at an older age. Milk supplementation of ARLb lambs resulted in improved coagulating ability of the rennet and enhanced cheese lipolysis after 60 d of ripening. A reduction of all free fatty acids was observed in all cheeses when passing from 20 to 40 d of slaughter of the lambs. Conjugated linoleic acids were more abundant in ARLb cheeses at both 20 and 40 d. Therefore, supplementation of the milk substitute with Lb. acidophilus improved the enzymatic features of rennet and the healthful and nutritional characteristics of it the ovine cheese. Moreover, the addition of lactobacilli to the milk substitute made it possible to increase the slaughter age of lambs without detrimental effects on rennet characteristics.