OBJECTIVES: Type 2 diabetes is partially caused by insufficient pancreatic β cell insulin secretion. Previous studies show therapeutic ultrasound (TUS) evokes insulin secretion from β cells as a potential treatment for type 2 diabetes; however, how β cells sense TUS and the broad effects of this treatment on cells remain unknown. Here, we identified mechanosensitive channels (MSC) expressed by β cells and TUS-mediated gene downregulation and reactive oxygen species (ROS) formation. METHODS: For all experiments, 1 W/cm(2) intensity and 800 kHz frequency TUS were continuously applied for 5 minutes with a 100% duty cycle. RNA and protein isolation of human pancreatic islets and the rat insulinoma INS 832/13 cell line were used for rtqPCR and western blot, respectively, to determine MSC expression. INS cells treated with MSC agonists and/or antagonists during TUS were visualized via fluorescent microscopy to track ROS formation. Using the same treatments, rtPCR analysis of INS insulin and IAPP encoding insulin and islet amyloid polypeptide (IAPP), respectively, was performed. TUS treatments were replicated in rats from which pancreatic sections were collected for immunohistochemistry analysis. RESULTS: We found the expression of TRPV2, TRPV5, and piezo1 in human islets and INS cells. TUS increased ROS formation in INS cells compared to sham-treated controls (P < .0001); however, modulation of MSC mitigated this effect (P < .001). TUS decreased the expression of the genes insulin and IAPP in INS cells compared to sham-treated controls (P < .001 and P < .01, respectively); however, complete MSC inhibition reversed this effect (P < .01 and P < .05, respectively). In our rat model, pancreatic and duodenal homeobox 1 (PDX1) expression was decreased by TUS compared to sham-treated controls (*P < .05); however, TUS did not decrease insulin or IAPP levels (P > .05). CONCLUSION: We report the expression of TRPV2, TRPV5, and piezo1 in human and rodent pancreatic β cells that are implicated in both TUS-mediated ROS formation and the downregulation of essential β cell genes.