Exocytosis is a crucial process of secreting various signaling molecules such as neurotransmitters, hormones, and other chemical mediators into the extracellular space Exocytotic release is caused by membrane fusion of intracellular vesicles with the plasma membrane triggered by an increase in intracellular Ca2+ In the present study, we developed an artificial system of exocytosis that secretes intravesicular contents upon Ca2+ influx We prepared artificial secretory cells using cell-sized giant unilamellar liposomal vesicles (GUVs) that contain small liposomes (SUVs) that correspond to secretory vesicles To observe exocytosis-like secretion in an at Diktat system, we labeled both an intra-SUV solution and an SUV membrane with a soluble fluorescent dye and a rhodamine-labeled phospholipid, respectively To induce membrane fusion between SUVs and a GUV as observed in exocytosis, the Ca2+ concentration of intra-GUV was elevated by incorporating ionomycin (a Ca2+ ionophore) into the GUV membrane We succeeded in inducing exocytosis-like secretion by Ca2+ elevation in a GUV together with the osmolarity difference between the intra-GUV and extra-GUV solutions