To explore the function of PPAR gamma in the goat mammary gland, we cloned the whole cDNA of the PPAR gamma gene. Homology alignments revealed that the goat PPAR gamma gene is conserved among goat, bovine, mouse, and human. Luciferase assays revealed that rosiglitazone enhanced the activity of the PPAR gamma response element (PPRE) in goat mammary epithelial cells (GMECs). After rosiglitazone (ROSI) treatment of GMECs, there was a significant (P < 0.05) increase in the expression of genes related to triacylglycerol synthesis and secretion: LPL, FASN, ACACA, PLIN3, FABP3, PLIN2, PNPLA2, NR1H3, SREBF1, and SCD. The decreases in expression observed after knockdown of PPAR gamma relative to the control group (Ad-NC) averaged 65%, 52%, 67%, 55%, 65%, 58%, 85%, 43%, 50%, and 24% for SCD, DGAT1, AGPAT6, SREBF1, ACACA, FASN, FABP3, SCAP, ATGL, and PLIN3, respectively. These results provide direct evidence that PPAR gamma plays a crucial role in regulating the triacylglycerol synthesis and secretion in goat mammary cells and underscore the functional importance of PPAR gamma in mammary gland tissue during lactation.