Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor (TF)-induced blood coagulation. A positive association between very low density lipoproteins (VLDLs) and TFPI has been reported in vivo. In contrast, one in vitro study indicates that TFPI may enhance lipoprotein lipase (LPL) activity, thereby increasing triglyceride hydrolysis. The current study was conducted to investigate how native VLDL influenced the synthesis and release of TFPI in endothelial cells, and how TFPI affected the LPL-induced hydrolysis of VLDL in vitro and at the endothelial surface. A spontaneously transformed immortal endothelial cell line (ECV304) and primary coronary artery cells (CoEc) were used, and VLDL was isolated from healthy volunteers by density gradient ultracentrifugation. Sequential free fatty acid (FFA) measurements were used to evaluate the kinetics of the LPL-induced hydrolysis. The levels of TFPI mRNAs in the stimulated cells were determined by quantitative real-time reverse transcription-ploymerase chain reaction (qPCR) using the ABI PRISM 7700 Sequence Detection System. Stimulation of ECV304 cells for 24 hours with native VLDL (0–100 μg/mL) caused a dose-dependent increase of TFPI in the medium (6.7–23.8 ng/10 6 cells, P < 0.001), without affecting the cellular content of TFPI. The expression of TFPI mRNA was significantly upregulated after 10 minutes of stimulation with n-VLDL. Both recombinant TFPI (r-TFPI) and LPL showed a dose-dependent binding to ECV 304 cells without saturation, and no competitive binding interactions between LPL and TFPI were observed at the endothelial surface. The addition of increasing concentrations of r-TFPI to ECV 304 cells, preincubated with LPL, did not affect the hydrolysis of VLDL triglycerides. The maximal reaction velocity (V max) of LPL-induced hydrolysis of n-VLDL was not affected by the addition of increasing concentrations of r-TFPI to the reaction mixture in vitro. The current experimental study indicates an upregulation of TFPI synthesis and release by VLDL. LPL-induced hydrolysis of VLDL in vitro was not influenced by TFPI neither in suspension nor at the endothelial surface.