A total of 30 Zamorano-type cheeses were manufactured in order to study the effects of milk conjugated linoleic acid (CLA) content, ripening time, and interactions between both effects on fatty acid (FA) profile. Cheeses elaborated from milk with a high CLA content showed higher contents of vaccenic, oleic, alpha-linolenic (ALA), CLA, monounsaturated (MUFA) and polyunsaturated (PUFA) FA, and lower contents of capric, lauric, myristic, palmitic, estearic, linoleic, and saturated (SFA) FA than cheese from milk with a low CLA content. Content of unsaturated C18 FA along with MUFA and PUFA groups increased throughout the ripening, while SFA content decreased. The interactions between milk CLA content and ripening time were significant for palmitic, linoleic, CLA, SFA, and omega-6/omega-3 ratio in Zamorano-type cheese. Decreases in cheese omega-6/omega-3 ratio were obtained from milk with a high CLA content; this ratio worsened over ripening in cheeses from low-CLA milk. In conclusion, these results emphasize the importance of the initial CLA content in milk with regard to improve the lipid profile in cheese for consumption.