Abnormal microRNA functions are closely associated with pancreatic β-cell loss and dysfunction in type 2 diabetes. Dysregulation of miR-30d has been reported in the individuals with diabetes. To study how miR-30d affects pancreatic β-cell functions, we generated two transgenic mouse lines that specifically overexpressed miR-30d in β-cells at distinct low and high levels. Transgenic overexpressed miR-30d systemically affected β-cell function. Elevated miR-30d at low-level (TgL, 2-fold) had mild effects on signaling pathways and displayed no significant changes to metabolic homeostasis. In contrast, transgenic mice with high-level of miR-30d expression (TgH, 12-fold) exhibited significant diet-induced hyperglycemia and β-cell dysfunction. In addition, loss of β-cell identity was invariably accompanied with increased insulin/glucagon-double positive bihormonal cells and excess plasma glucagon levels. The transcriptomic analysis revealed that miR-30d overexpression inhibited β-cell-enriched gene expression and induced α-cell-enriched gene expression. These findings implicate that an appropriate miR-30d level is essential in maintaining normal β-cell identity and function.