The PPARγ is a ligand-dependent transcription factor coordinating lipogenic genes in the mammary gland and can be modified by conjugated linoleic acid (CLA) and/or chemical agonists. This study used the PPARγ agonist Tiazolinediona (TZD) to evaluate the effect on lipogenic gene expression. Mammary explants were cultured in vitro for 24h with the following treatments: (1) Control: 400µL of mammary epitelial growth medium; (2) TZD: Control + 40 µL of TZD (10 µmol/L); (3) CLA: Control + 30 µmol CLA (315 µmol/L) and (4) TZDCLA: Control + TZD (10 µmol/L) + 30 µmol CLA (315 µmol/L). The CLA used was a mixture 50:50 of cis-9,trans-11 and trans-10,cis-12. The RNA was extracted, complementary DNA (cDNA) synthesized and qRT-PCR carried out, measuring the gene expression of acetyl-CoA-carboxylase α (ACCα transcript from promoter III), fatty acid synthase (FASN), peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element binding protein-1 (SREBP1), sterol regulatory element-binding protein cleavage-activating protein (SCAP), stearoyl CoA desaturase (SCD), insulin-induced gene 1 (INSIG1), insulin-induced gene 2 (INSIG2). The data were analyzed using the PROC MIXED using treatment and sample as fixed effects and the geometric mean of ribossomal protein 18 (S18) and β-actin as a covariate. Compared with Control, TZD treatment increased the gene expression of SREBP1 (P = 0.0001), INSIG1 (P = 0.0001), INSIG2 (P = 0.005), FASN (P = 0.0001), ACCα (P = 0.0001), SCD1 (P = 0.0001) e PPARγ (P = 0.0001) in 10.1-, 7.9-, 8.5-, 78.3-, 87.5-, 62.7-, and 6.2-fold, respectively. The CLA compared with Control, decreased the expression of FASN (P = 0.04), SCD (P = 0.01) e ACCα (P = 0.05), in 2.8-, 2.1- and 0.4-fold, respectively. Comparing TZDCLA and CLA treatments, TZDCLA decreased the expression of FASN (P = 0.01) and INSIG2 (P = 0.0005) in 2.7 and 15.7 fold, respectively. Overall, our results showed a positive and consistent effect of TZD increasing the gene expression of PPARγ and its targeted genes and CLA reducing the expression of genes involved in milk fat depression.