MicroRNAs have emerged as essential regulators of beta cell function and beta cell proliferation. One of these microRNAs, miR-132, is highly induced in several obesity models and increased expression of miR-132 in vitro modulates glucose-stimulated insulin secretion. The aim of this study was to investigate the therapeutic benefits of miR-132 overexpression on beta cell function in vivo. To overexpress miR-132 specifically in beta cells, we employed adeno-associated virus (AAV8)-mediated gene transfer using the rat insulin promoter in a double-stranded, self-complementary AAV vector to overexpress miR-132. Treatment of mice with dsAAV8-RIP-mir132 increased miR-132 expression in beta cells without impacting expression of miR-212 or miR-375. Surprisingly, overexpression of miR-132 did not impact glucose homeostasis in chow-fed animals. Overexpression of miR-132 did improve insulin secretion and hence glucose homeostasis in high-fat diet-fed mice. Furthermore, miR-132 overexpression increased beta cell proliferation in mice fed a high-fat diet. In conclusion, our data show that AAV8-mediated gene transfer of miR-132 to beta cells improves beta cell function in mice in response to a high-fat diet. This suggests that increased miR-132 expression is beneficial for beta cell function during hyperglycemia and obesity.