Two experiments were conducted to evaluate the effects of nature of forage on fatty acid composition and lipolytic system in cow milk to increase the nutritional quality of dairy products. Each experiment was divided into a 4-wk preexperimental and 6- or 8-wk experimental period. During the 2 preexperimental periods, 56 midlactating Montbeliarde or Tarentaise cows received a diet based on corn silage. Subsequently, in Experiment 1, 40 cows were allocated into 5 groups (4 Montbeliarde and 4 Tarentaise cows per group) and assigned to dietary treatments: corn silage (87% of dry matter intake), grass silage (86%), ryegrass hay (90%), mountain natural grassland hay (87%), or a diet rich in concentrate (CONC, 65/35% concentrate/hay). In Experiment 2, 16 cows divided into 2 groups were fed during 3 or 6 wk mountain natural pasture (100%) or mountain natural grassland hay (87%). Principal component analysis was applied to describe the relationships among dairy performances, milk fatty acids (FA), and lipolytic system. The milk 18: 0, cis-9-18:1, trans-11-18:1, and cis-9, trans-11-18:2 percentages were closely associated with 3-wk mountain natural pasture diet, whereas short- and medium-chain (mostly saturated) FA were associated with the CONC diet. Tarentaise milk fat contained a lower proportion (-3 to 4 g/100 g) of 16:0 and higher proportions of stearic acid and fewer markedly polyunsaturated FA than Montbeliarde milk fat. Milk lipolysis was lowest for CONC and corn silage groups. Milk from Tarentaise cows presented lower initial free FA and postmilking lipolysis. Diets given to cows, especially young grass, modified the milk content of FA with a putative nutritional effect on human health.