Vascular endothelial cells (ECs) are constantly subjected to rhythmic distension because of pulsatile flow in the circulation. The rhythmic distension-induced cyclic strain plays an important role in modulating endothelial physiology. In the present study, we investigated the effects of cyclic strain on the expression of thrombomodulin (TM), an anti-coagulant protein that possesses anti-inflammation properties. Exposure of ECs to cyclic strain increased NADH/NADPH oxidase and endothelial nitric oxide synthase (eNOS) activities, as well as the nitric oxide (NO) production. When ECs were subjected to a greater cyclic strain (21%), TM expression was increased. The increased TM expression was not observed when ECs were subjected to a lower cyclic strain (15%). ECs treated with an NO donor (NOC18), induced TM expression, whereas hydrogen peroxide (H2O2) treatment did not have significant effects on TM expression. Pretreatment of ECs with an eNOS inhibitor (L-NAME) abolished the cyclic strain-induced TM expression in ECs. These results suggest that cyclic strain to ECs induces NO production which consequently results in an increase of TM expression in ECs. Our findings provide insights into the mechanisms by which cyclic strain induces TM expression in ECs, therefore playing important roles in anticoagulation and atheroprotection in the vascular system.