The influence of three probiotic strains ( Lactobacillus acidophilus, Lactobacillus paracasei and Bifidobacterium lactis) in semi-hard cheese proteolysis patterns was assessed. Probiotics were inoculated both as single cultures and as a three-strain mix, and added to milk either after a pre-incubation step or directly to the vat. B. lactis did not show any effect on proteolysis of cheeses, while L. paracasei showed limited impact at the end of the ripening. In contrast, L. acidophilus significantly influenced secondary proteolysis from the beginning of ripening, causing an increase in the levels of small nitrogen-containing compounds and free amino acids and changes in the peptide profiles. The effect of Lactobacillus acidophilus on peptidolysis was more noticeable when it was added to cheese–milk after pre-incubation in an enriched milk fat substrate. Similar results obtained with the three-strain mixed culture, suggesting that L. acidophilus played a major role in secondary proteolysis of probiotic cheeses in this trial.