Actin has long been known to be involved with exocytosis in a variety of cell systems. However, the mechanisms of actin remodelling remain unclear. To better understand actin dynamics, we used live-cell two-photon imaging of mouse beta cells isolated from transgenic mouse expressing Lifeact-eGFP; a fluorescent probe that has a low affinity for filamentous (F-actin). Simultaneously we recorded glucose-induced insulin granule fusion using the entry of Sulforhodamine B (SRB) extracellular dye into each granule as it fused with the cell membrane. Our data showed that each granule fusion event was associated with a transient increase in F-actin at the site of fusion. Application of 100μM Latrunculin B, which sequesters actin monomers, enhanced the number of glucose-induced fusion events but abolished the F-actin increase. This suggests F-actin remodelling, at the sites of fusion, is by nucleation rather than movement. Inhibition of actin nucleator Arp2/3-complex with 100μM CK666 blocked F-actin changes at the sites of insulin granule fusion and slowed the decay time of SRB fluorescence, indicating altered granule fusion kinetics. The secretory phenotype was further examined using insulin secretion assay. CK666 treated primary mouse beta cells showed a significantly reduced glucose stimulated insulin secretion compared with control conditions. The data suggests Arp2/3-mediated actin nucleation plays important role in facilitating insulin granule fusion. Western blot and qPCR data confirmed Arp2/3 is present in mouse pancreatic islets and MIN6 cells. To further validate Arp2/3’s function in insulin secretion, shRNA Knock-down (KD) approach was used to KD Arp3 in MIN6 cells. The Arp3-KD MIN6 cells showed a significant decrease of Apr3 protein level and correspondently reduced glucose induced insulin secretion. Together, these results provide evidence that F-actin is dynamically remodelled by Arp2/3 nucleation at the sites of insulin granule fusion and this plays an essential role in efficient secretion.