A direct comparison of cow and goat performance and milk fatty acid (FA) responses to diets that either induce milk fat depression or increase milk fat content in cows suggests species-specific regulation of lipid metabolism, including mammary lipogenesis. This experiment was conducted to highlight potential mechanisms responsible for the differences in mammary lipogenesis due to diet and ruminant species. Twelve Holstein cows and 12 Alpine goats were fed a basal diet containing no additional lipid (CTL) or a similar diet supplemented with corn oil [5% dry matter intake (DMI)] and wheat starch (COS), marine algae powder (MAP; 1.5% DMI), or hydrogenated palm oil (HPO; 3% DMI), according to a 4 × 4 Latin square design with 28-d experimental periods. Milk yield, milk composition, FA profile, and secretions were measured. On d 27 of each experimental period, the mRNA abundance of 21 genes involved in lipid metabolism or enzyme activities or both were measured in mammary tissue sampled by biopsy. The results showed significant differences in the milk fat response of cows and goats to the dietary treatments. In cows, fat content was lowered by COS (-45%) and MAP (-22%) and increased by HPO (+13%) compared with CTL, and in goats only MAP had an effect compared with CTL, with a decrease of 15%. In both species, COS and MAP lowered the yields (mmol/d per kilogram of body weight) of <C16 and C16 FA. With COS, this decrease was compensated by an increase of >C16 FA in goats but not in cows, and the >C16 FA yield decreased with MAP in both species. Supplementation of HPO increased the yield of milk C16 FA (mmol/d per kilogram of body weight) in cows. These variations in milk fat content and FA secretion were not associated with modifications in the mammary expression of 21 genes involved in major lipid pathways, except for 3 transcription factors: PPARA, INSIG1, and SP1. This absence of large changes might be due to post-transcriptional regulation of these genes and related to the time of sampling of the mammary tissue relative to the previous meal and milking or to differences in the availability of substrate for the corresponding proteins. However, the abundance of 14 mRNA among the 21 encoding for genes studied in the mammary gland was significantly different among species, with 5 more abundant in cows (FADS3, ACSL1, PPARA, LXRA, and PPARG1) and 10 more abundant in goats (FASN, CD36, FABP3, LPL, GPAM, LPIN1, CSN2, MFGE8, and INSIG1). These species specificities of mammary lipid metabolism require further investigation.