Insulinoma is the main type of functional pancreatic neuroendocrine tumors. The functional microRNAs (miRNAs) regulating tumor growth and progression in insulinomas are still unknown. We conducted the miRNA expression profile analysis using miRNA quantitative RT-PCR array and identified 114 differentially expressed miRNAs in human insulinomas compared with normal pancreatic islets. Forty-one differentially expressed miRNAs belonged to 7 miRNA families, and 28 miRNAs in 3 of the families localized in the epigenetically regulated imprinted chromosome 14q32 region. We validated the most significant differentially expressed miRNA cluster miR-144/451 in another 8 human normal islet samples and 25 insulinomas. Our data showed that the overexpression of miR-144/451 in mouse pancreatic β-cells promoted cell proliferation by targeting the β-cell regulator phosphatase and tensin homolog deleted on chromosome ten/v-akt murine thymoma viral oncogene homolog pathway and cyclin-dependent kinase inhibitor 2D. Our findings highlight the importance of functional miRNAs in insulinomas.