© 2015, eLife Sciences Publications Ltd. All rights reserved. It is unclear that how subcellular organelles respond to external mechanical stimuli. Here, we investigated the molecular mechanisms by which mechanical force regulatesCa2+signaling at endoplasmic reticulum (ER) in human mesenchymal stem cells. Without extracellularCa2+, ERCa2+release is the source of intracellularCa2+oscillations induced by laser-tweezer-traction at the plasma membrane, providing a model to study how mechanical stimuli can be transmitted deep inside the cell body. This ERCa2+release upon mechanical stimulation is mediated not only by the mechanical support of cytoskeleton and actomyosin contractility, but also by mechanosensitive Ca2+permeable channels on the plasma membrane, specifically TRPM7. However,Ca2+influx at the plasma membrane via mechanosensitiveCa2+permeable channels is only mediated by the passive cytoskeletal structure but not active actomyosin contractility. Thus, active actomyosin contractility is essential for the response of ER to the external mechanical stimuli, distinct from the mechanical regulation at the plasma membrane.