Abstract: Endothelial cells (ECs) regulate vascular function by converting mechanical forces into biochemical signals; however, the molecular mechanisms of pN-scale mechanotransduction remain elusive. Here, we develop an optical tweezer-integrated confocal microscopy system that allows precise, noninvasive manipulation of the cell membrane localization with mechanical stimuli within the 0–100 pN range while monitoring Ca2+-mediated NO/ROS redox signaling in situ in single ECs under varying force parameters. We show that pN-scale mechanical stimulation regulates extracellular Ca2+ influx, triggering downstream production of NO and ROS, which subsequently affects intracellular redox homeostasis. Key mechanosensitive ion channels (e.g., Piezo1 and TRPV4) and cytoskeletal components (e.g., F-actin) facilitate force-induced redox signaling. We further delineate the roles of membrane tension-dominant versus hybrid tension-tether models in mechanotransduction, revealing their differential engagement in force transmission pathways. This mechanistic framework establishes direct connections between pN-scale mechanical input characteristics and redox-regulated vascular homeostasis.