Stearic acid (C18:0) has shown be able to increase milk fat in ruminants but the results are variable. This study evaluated the effect of feeding stearic acid (C18:0) and its interaction with CLA trans-10,cis-12 on the transcription of genes involved in milk fat synthesis in ewes. Twenty-eight Lacaune ewes (36 ± 2 DIM; 73 ± 9 kg BW) were randomly assigned to one of the following treatments (7/treatment) for 21 d (7 d for adaptation and 14 d of data collection): 1) Control (no CLA or C18:0); 2) C18:0 (32 g/d, 87%); 3) CLA (27 g/d orally dosed containing 29.9% trans-10,cis-12 CLA,); and 4) CLAC18:0. Mammary biopsies were taken at d 14 of data collection, RNA was extracted, cDNA synthesized and RT-qPCR analysis conducted for ACACAα PII, FASN, SCD, LPL, CD36, FABP (3 and 4). The data were analyzed using the MIXED procedure of SAS using treatment as fixed effect and animal as random. The geometric mean of the housekeeping genes (S18 and actin-β) were used to normalize the gene expression. Compared with Control, C18:0, CLA and CLAC18:0 reduced the expression of ACACAα PII in 1.8-, 2.3- and 1.4-fold (P = 0.01 for all), respectively, and CLA reduced expression by 1.6-fold (P = 0.01) compared with CLAC18:0. Compared with Control, CLA reduced FASN and LPL by 1.7-fold (P = 0.02) and CLAC18:0 reduced LPL expression by 1.3-fold (P = 0.05). Expression of CD36 in C18:0 was increased, compared with Control and CLAC18 in 1.8-fold (P = 0.05) and 2.4-fold (P = 0.01), respectively. SCD expression was, respectively, decreased in 1.6-, 1.6- and 1.6-fold (P = 0.01 for all) in C18:0, CLA and CLAC18:0 when compared with Control. The FABP4 was 2.1-fold (P = 0.01) higher in C18:0 compared with CLAC18:0. Overall, C18:0 was not able to overcome the downregulating effect of CLA trans-10,cis-12 on mammary lipogenic genes when combined with C18:0