During lactation the mammary gland produces a substantial amount of triglycerides using fatty acids synthesized in the mammary gland and from the plasma, prioritizing milk fat synthesis over adipose tissue, especially at the beginning of lactation. Specific fatty acid transporter proteins and enzymes are involved in fatty acid uptake by mammary cells and triglyceride synthesis. The objective of this study was evaluate gene expression of long chain acyl-CoA synthetase (ACSL1), solute carrier family fatty acid transporter (SLC27A6), fatty acid binding proteins (FABP3 and FABP4), fatty acid translocator CD36 (FATCD36), lipoprotein lipase (LPL), acylglycerol phosphate acyltransferase (AGPAT6), lipin (LIPIN1), diacylglycerol acyltransferase (DGAT1), and peroxisome proliferator-activated receptor gamma (PPARγ) at different stages of lactation in dairy ewes. Mammary gland biopsies were taken from 6 lactating ewes at 15, 70, and 120 DIM, to represent early, mid, and late lactation. Total RNA was extracted, cDNA synthesized and quantitative real-time PCR analysis conducted. Data were analyzed by PROC MIXED (SAS Institute) procedure using stage of lactation as a fixed effect, animal as random, and the geometric mean of the housekeeping genes (ribosomal protein S18 and β-actin) as a covariate. Data points with Studentized residuals outside of ± 2.5 were considered outliers and excluded from analysis. There was not effect of stage of lactation for ACSL1, SLC27A6, and FABP4 transcripts (P > 0.05). The expression of FABP3 and FATCD36 was higher in early lactation and decreased as lactation progressed (P < 0.05). Similarly, the transcripts of AGPAT6 and DGAT1 were higher in early lactation (P < 0.05) and LIPIN1 tended to be increased in early lactation (P = 0.09). In addition, LPL and PPARγ were increased in early lactation compared with mid and late lactation (LPL P = 0.01 and P = 0.002 and PPARγ P = 0.03 and P = 0.03, respectively). Our results show a higher expression of fatty acid transporters and key enzymes in mammary tissue at early stages of lactation prioritizing milk fat synthesis.