A direct comparison of cow and goat performance and milk fatty acid (FA) responses to diets known to induce milk fat depression in the bovine has suggested interspecies differences in rumen and mammary lipid metabolism. Thus, this study was conducted to infer some potential mechanisms responsible for the differences in mammary lipogenesis due to diet and ruminant species. To meet this objective, 12 cows and 15 goats were fed a basal diet (control), a similar diet supplemented with 2.2% fish oil (FO), or a diet containing 5.3% sunflower oil and additional starch (+38%; SOS) according to a 3 × 3 Latin square design with 26-d experimental periods. Milk yield, milk composition, FA profile, and FA secretion were measured. On the last day of each period, the mRNA abundance of 19 key genes in mammary metabolism or the enzyme activity or both were measured in mammary tissue sampled by biopsy or at slaughter or both. The results show significant differences in the response of cows and goats to the dietary treatments. In cows, milk fat content and yield were lowered by FO and SOS (−31%), whereas only FO decreased milk fat content in goats (−21%) compared with the control. In cows and to a lesser extent in goats, FO and SOS decreased the secretion of C16 FA output (mmol/kg of BW). However, SOS increased the secretion of >C16 FA in goats. These changes in milk fat content and FA secretion were not associated with modifications in mammary expression or the activity of 19 proteins involved in the major lipogenic pathways. This absence of variation may be attributable to posttranscriptional regulation for these genes or related to the time of sampling of the mammary tissue relative to the previous meal and milking. Otherwise, the abundances of 15 mRNA among the 19 encoding for genes involved in lipid metabolism in the mammary gland were different among species, with 9 more abundant in cows (FASN, FADS1, SCD1, GPD1, LALBA, SREBF1, LXRA, PPARA, and PPARG1) and 6 more abundant in goats (G6PD, GPAM, SCD5, XDH, CSN2, and SP1). Similarly, a significant effect of the species was observed in the 4 enzyme activities measured; glycerol-3-phosphate dehydrogenase and malic enzyme were higher in cows, and FA synthase and glucose-6-phosphate dehydrogenase activities were higher in goats. In conclusion, the differences between cow and goat performance and milk FA responses to the FO and SOS treatments were not related to changes in the measured mammary lipogenic gene expression. Furthermore, the data provide evidence that the major mammary lipogenic pathways differ between the caprine and the bovine, whose biological significance remains to be unraveled.