Milk fat synthesis involves biochemical processes, including fatty acid synthesis, uptake, transport and desaturation. Trans-10,cis-12 CLA inhibits milk fat synthesis by decreasing the expression of genes and transcription factors. Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of lipid synthesis in many tissues and is affected by trans-10,cis-12 CLA but, the mechanisms by which trans-10,cis-12 CLA suppresses the expression or activity of PPARγ and expression of its targeted genes are not clear. This study used a chemical PPARγ agonist to evaluate the effect of PPARγ on mammary lipid synthesis and its interaction with trans-10,cis-12 CLA in lactating ewes. Twenty-four crossbred lactating ewes [70 ± 3 DIM; 60 ± 0.45 kg body weight (BW)] were randomly assigned to one of the 4 treatments (n = 6/treatment) for 7 d. Treatments were (1) Control (intravenous infusion of 100 mL/d of saline); (2) TZD (intravenous infusion of 4 mg/kg of BW per d in 100 mL of saline); (3) CLA (27 g/d orally dosed methyl ester containing 29.9% trans-10,cis-12 CLA); and (4) TZD+CLA. Mammary and adipose tissue biopsies were taken, RNA was extracted, cDNA synthesized and qRT-PCR analysis conducted for PPARγ, SREBP1 and SCD1. Compared with control, fat content was 22.3% lower in CLA (P = 0.05), tended to be 20.7% lower in TZD+CLA (P = 0.06). In the mammary gland, CLA decreased expression of PPARγ, SREBP1 and SCD1 by 64.4, 60 and 19% compared with control (P = 0.02, P = 0.01 and P = 0.005, respectively), confirming its negative effects on the expression of lipogenic genes. However, TZD did not stimulate the expression of these genes or overcome the effect of CLA in mammary tissue. In adipose tissue, expression of PPARγ were not affected by treatment, whereas SREBP1 expression was increased by TZD, CLA and TZD+CLA compared with control and SCD1 expression was higher in TZD+CLA compared with the other treatments. Overall, CLA negatively affected mammary expression of genes involved in lipid synthesis and TZD was unable to overcome those effects demonstrating that the mechanism of CLA is not dependent on inhibition of PPARγ