Key points Comprehensive imaging analyses of glucose transporter 4 (GLUT4) behaviour in mouse skeletal muscle was conducted. Quantum dot‐based single molecule nanometry revealed that GLUT4 molecules in skeletal myofibres are governed by regulatory systems involving ‘static retention’ and ‘stimulus‐dependent liberation’. Vital imaging analyses and super‐resolution microscopy‐based morphometry demonstrated that insulin liberates the GLUT4 molecule from its static state by triggering acute heterotypic endomembrane fusion arising from the very small GLUT4‐containing vesicles in skeletal myofibres. Prior exposure to exercise‐mimetic stimuli potentiated this insulin‐responsive endomembrane fusion event involving GLUT4‐containing vesicles, suggesting that this endomembranous regulation process is a potential site related to the effects of exercise. Abstract Skeletal muscle is the major systemic glucose disposal site. Both insulin and exercise facilitate translocation of the glucose transporter glucose transporter 4 (GLUT4) via distinct signalling pathways and exercise also enhances insulin sensitivity. However, the trafficking mechanisms controlling GLUT4 mobilization in skeletal muscle remain poorly understood as a resuly of technical limitations. In the present study, which employs various imaging techniques on isolated skeletal myofibres, we show that one of the initial triggers of insulin‐induced GLUT4 translocation is heterotypic endomembrane fusion arising from very small static GLUT4‐containing vesicles with a subset of transferrin receptor‐containing endosomes. Importantly, pretreatment with exercise‐mimetic stimuli potentiated the susceptibility to insulin responsiveness, as indicated by these acute endomembranous activities. We also found that AS160 exhibited stripe‐like localization close to sarcomeric α‐actinin and that insulin induced a reduction of the stripe‐like localization accompanying changes in its detergent solubility. The results of the present study thus provide a conceptual framework indicating that GLUT4 protein trafficking via heterotypic fusion is a critical feature of GLUT4 translocation in skeletal muscles and also suggest that the efficacy of the endomembranous fusion process in response to insulin is involved in the benefits of exercise.