The Coptis chinensis (Franch) is widely used in diabetes therapies in traditional folk medicine from China, and we previously reported that its main active component, berberine (BBR), acted as an insulin secretagogue through blocking the KCNH6 potassium channel. However, the specific actions of BBR on insulin secretory granule (ISG) dynamics are largely unknown. Here, we analyzed the docking and fusion of ISGs from beta-cells exposed to either short-term or long-term treatment with BBR. Under short-term treatment, at 8.3 mmol/L glucose, BBR slightly induced insulin secretion with a gradually increasing second phase only, showing an increased number of ISGs fused without stable docking to the plasma membrane. However, in the presence of 16.7 mmol/L glucose, biphasic insulin secretion by BBR was augmented significantly. The intracellular Ca2+ level increased during the second phase by BBR, suggesting that the Ca2+ dynamics contribute to the dynamics of insulin exocytosis. Under long-term BBR treatment for db/db mice, BBR restored impaired biphasic phases of insulin secretion by recovering the number of ISGs fused with or without predocking to the plasma membrane. In addition, BBR enhanced the docking capacity and increased biphasic Ca2+ concentration after glucose stimulation. Further research revealed that the short-term treatment with BBR primarily promoted the fusion of ISGs through blocking KCNH6 channels, whereas the long-term treatment with BBR improved the docking and fusion of ISGs by an additional effect on activating the cAMP-PKA-CREB pathway. Hence, our study indicated that short-term and long-term treatment with BBR promoted insulin exocytosis through different mechanisms in pancreatic beta cells. BBR could be a dual-action antidiabetic agent, acutely enhancing insulin secretion in response to glucose and chronically improving beta-cell function in T2D.