As a valuable nutrient in milk, fat accounts for a significant proportion of the energy requirements of ruminants and is largely responsible for determining milk quality. Fatty acids (FAs) are a pivotal component of milk fat. Conjugated linoleic acid (CLA) is one of the naturally occurring FAs prevalent in ruminant dairy products and meat. Increasing attention has been given to CLA because of its anti-cancer, anti-inflammatory, immune regulation, and lipid metabolism regulation properties, and these benefits potentially contribute to the growth and health of infants. In breast milk, CLA is present in trace amounts, mainly in the form of cis-9, trans-11 CLA. Notably, cis-9, trans-11 CLA improves the milk fat rate while trans-10, cis-12 CLA inhibits it. Apart from having multiple physiological functions, CLA is also a pivotal factor in determining the milk quality of ruminants, especially milk fat rate. In response to growing interest in green and healthy functional foods, more and more researchers are exploring the potential of CLA to improve the production performance of animals and the nutritional value of livestock products. Taken together, it is novel and worthwhile to investigate how CLA regulates milk fat synthesis. It is the purpose of this review to clarify the necessity for studying CLA in ruminant milk fat and breast milk fat.