During high tidal volume mechanical ventilation in patients with acute lung injury (ALI)/ARDS, regions of the lung are exposed to excessive stretch, which causes inflammatory responses and further lung damages. In this study, the effects of mechanical stretch on intracellular Ca²⁺ concentration ([Ca²⁺]_i), which regulates a variety of endothelial properties, were investigated in human pulmonary microvascular endothelial cells (HPMVECs). HPMVECs grown on fibronectin-coated silicon chambers were exposed to uni-axial stretching using a cell stretching apparatus. Following stretching and subsequent unloading, [Ca²⁺]_i measured by fura-2 fluorescence was transiently increased in a strain amplitude-dependent manner. The [Ca²⁺]_i elevation induced by stretch was not observed in the Ca²⁺-free solution and was blocked by Gd³⁺, a stretch-activated channel inhibitor, or ruthenium red, a TRPV inhibitor. Disruption of actin polymerization with cytochalasin D inhibited the stretch-induced [Ca²⁺]_i elevation. In contrast, increases in [Ca²⁺]_i induced by thapsigargin or thrombin were not affected by cytochalasin D. Increased actin polymerization with sphingosine-1-phosphate or jasplakinolide enhanced the stretch-induced [Ca²⁺]_i elevation. A simple network model of the cytoskeleton was also developed which supports the notion that actin stress fibers are required for efficient force transmission to open stretch-activated Ca²⁺ channels. In conclusion, mechanical stretch activates Ca²⁺ influx via stretch-activated channels different from other Ca²⁺ influx pathways such as receptor- and store-operated Ca²⁺ entries in HPMVECs that is tightly regulated by the actin cytoskeleton. These results suggest that abnormal Ca²⁺ homeostasis due to excessive mechanical stretch during mechanical ventilation may play a role in ALI/ARDS progression.