Long-term mammary expression patterns of lipogenic gene networks due to dietary lipid remain largely unknown. Mammary tissue was biopsied for transcript profiling of 29 genes at 0, 7, and 21 days of feeding cows saturated lipid (EB100) or a blend of fish/soybean oil (FSO) to depress milk fat. Milk fat yield decreased gradually with FSO and coincided with lower molar yield of fatty acids synthesized de novo, stearic acid, and oleic acid. The PPARγ targets LPIN1 and SREBF1 along with ACSS2, ACACA, FASN, and LPL increased by day 7 of feeding EB100, but differences between diets disappeared by day 21. Expression of SCAP increased markedly over time with FSO and differed from EB100 by approximately sevenfold on day 21. Expression of THRSP decreased by day 7 with both diets and returned to basal levels by day 21. SCD expression increased linearly through 7 days and remained elevated with both diets, a likely mechanism to ensure the proper level of endogenous oleic acid via desaturation of dietary stearate (EB100) or via more SCD protein to account for the reduction in stearate supply from the rumen (FSO). Despite this response, endogenous oleate was insufficient to restore normal milk fat synthesis. Only 2 of 29 genes differed in expression between diets on day 21, suggesting that transcriptional control mechanisms regulating fat synthesis were established as early as 7 days post-feeding. Gene expression reflected vastly different physiological responses by mammary tissue to adjust its metabolism to the influx of saturated fatty acids, trans10-18:1, and/or to the lack of stearic acid.