Increased plasminogen activator inhibitor-1 (PAI-1) level is the risk of thrombotic disease and atherosclerosis (As). Our research aims to study the effect and mechanism of PPARδ antagonist GW501516 on the expression of PAI-1. Firstly, human umbilical vein endothelial cells (HUVECs) were incubated with DMEM, and then treated with siRNA and TGFβ-Smad3 inhibitor, respectively. The protein and mRNA expression were examined by Western blotting assays and Real-time quantitative PCR, respectively. The result showed that GW501516 induced PAI-1 mRNA and protein expression in HUVECs compared with control groups (P < 0.05). According to PPARδ gene, the designed PPARδ siRNA primer silenced the PPARδ expression and depressed the induction of GW501516 on PAI-1 expression. Then, the HUVECs were treated by SB431542 or SIS3, which is the TGFβ-Smad3 signaling pathway blocker, and found that PAI-1 expression of cells were down-regulated.At last, we found that SB431542, SIS3 and GW501516 inhibited the expressions of pSmad3 protein. These results suggested that TGF beta-Smad3 signaling pathway involved in the regulation of GW501516-induced PAI-1 expression in HUVECs.