Actin bundles in vascular endothelial cells (ECs) play a critical role in transmitting intracellular forces between separate focal adhesion sites. However, quantitative descriptions of tension level in single actin bundles in a physiological condition are still poorly studied. Here, we evaluated magnitude of preexisting tension in a single actin bundle of ECs on the basis of measurements of its preexisting stretching strain and tensile properties. Cultured ECs expressing fluorescently-labeled actin were treated with detergents to extract acin bundles. One end of an actin bundle was then dislodged from the substrate by using a microneedle, resulting in a shortening of the actin bundle due to a release of preexisting tension. Assuming the shortened actin bundle reached its non-stress state, preexisting stretching strain was determined to be 0.24 on average. A tensile test of the dislodged single acin bundle was conducted with a pair of cantilevers to measure the force required for stretching it up to the original length, yielding an estimate of preexisting tension in the actin bundle. The magnitude of the preexisting tension, 4 nN on average, was comparable to previously reported data of the traction force generated by adherent cells at single adhesion sites to keep cell integrity. The Young's modulus of the isolated actin bundle was estimated to be approximately 300 kPa from the tensile tests together with evaluation of average diameter of the isolated actin bundle based on transmission electron microscopy. These data will contribute to better understanding of intracellular stress transmission mechanism in ECs.