Introduction: Elevated cyclic strain often leads to vein graft rejection, which is correlated to increased thrombomodulin release. Limited in vitro and in vivo studies document this release but the pathway involved is still unclear. In this study, we examine thrombomodulin (TM) expression and release after elevated cyclic strain. We also look at how physiological levels of shear stress impact TM expression and release. We assess the effect that various inflammatory mediators (i.e., ox-LDL) have on this cyclic strain-induced TM response. We investigate a range of signalling components (i.e., eNOS) in the cyclic strain-mediated TM expression and release pathway. We finish on the possible microvesicle (MV) involvement in cyclic strain- induced TM release. Hypothesis: Thrombomodulin (TM), an integral membrane protein involved in coagulation, is regulated by cyclic strain and plays a pivotal role in endothelial dysfunction. Results: (i) Following cyclic strain (7.5%), TM expression was down-regulated in both protein and RNA but up-regulated release into the cell culture media. (ii) Physiological levels of shear stress (10 dynes/cm2) acted as a more potent stimulus for TM release (when compared to cyclic strain) and also increased TM mRNA expression. (iii) Both TNF-α and ox-LDL had a down-regulatory effect on TM expression in static HAECs whereas glucose has no effect. In addition, TNF-α or ox-LDL caused an additive increase in cyclic strain-induced TM release. By contrast, glucose was found to “attenuate” cyclic strain-mediated TM release. (iv) We established that inhibition of PTK and p38 MAPK increased cyclic strain-mediated TM expression (i.e., reverses the typical strain-dependent response), thus play are role in the cyclic strain-decrease TM pathway. (v) We showed that MMPs and ROS decreased cyclic strain-induced TM release (i.e., reverses the typical strain-dependent response), thus they are involved in the cyclic strain-induced TM release pathway. (vi) We also observed that inhibition of integrin ανβ3 causes an additional increase in TM release in both static and strained HAECs, suggesting that integrins serve as a cellular “brake” for the release of TM. (vii) We noted that cyclic strain-induced TM release has some microvesicle involvement with one third of the release on exosomes. Conclusions: Cyclic strain regulates the release and expression of thrombomodulin in endothelial cells. TM release is regulated by MMPs and ROS, although a portion of release is due to microvesicle involvement.