The acoustic radiation forces produced by ultrasonic stimulation induce shear stress on objects in the acoustic field. Piezo1, a mechanosensitive ion channel protein that is expressed on the plasma membranes of vertebrate cells, can sense shear stress and transduce it into downstream signaling. In this study, we examined the sensitivity of Piezo1 to ultrasonic stimulation and assessed its downstream biological functions in human umbilical vein endothelial cells (HUVECs). Ultrasonic stimulation using a stimulation power of 0.2 W and a frequency of 1 MHz for 10 s did not induce cell damage. However, ultrasonic stimulation induced an influx of calcium ions, which increased with an increase in the stimulation duration. Knockdown of Piezo1 protein decreased the influx of calcium ions during ultrasonic stimulation, which demonstrated that Piezo1 may be activated by the shear stress produced by ultrasonic stimulation. The influx of calcium ions in response to ultrasonic stimulation could be modulated by the Piezo1 protein level. Additionally, ultrasonic stimulation reduced the levels of downstream factors such as MLCK and ATP, which are involved in the Ca²⁺/CaM/MLCK pathway, by suppressing Piezo1. As the Ca²⁺/CaM/MLCK pathway influences the permeability of the cell membrane, the internalization of FITC-Dextran into cells under ultrasonic stimulation was validated. Ultrasonic stimulation was demonstrated to promote the increase in cell permeability, and the suppression of Piezo1 was shown to induce the decrease in cell permeability. Therefore, this study shows that ultrasonic stimulation may modulate the permeability of the membrane of HUVECs by modulating the expression of Piezo1 protein.