Ionomycin is a calcium ionophore that induces release of calcium ions (Ca²⁺) from cellular storage to cytoplasm and Ca²⁺ influx from the outside of the cell. We investigated the effect of ionomycin on endoplasmic reticulum (ER)-Golgi transport in the vesicular stomatitis virus glycoprotein (VSV-G) system. Ionomycin inhibited transport of VSV-G in a concentration-dependent manner in baby hamster kidney (BHK) cells and HeLa cells. Half-maximum inhibition was observed at 5 μM. The inhibitory effect of ionomycin was not dependent on the cytoplasmic portion. Chelation of Ca²⁺ in culture medium did not affect transport efficiency, but co-incubation with ionomycin completely shut off transport. These findings highlight the importance of Ca²⁺ release from cellular storage. Because the inhibitory effect of ionomycin was expected to be dependent on mutual interaction of VSV-G and the ER chaperone calnexin, we further investigated interaction kinetics. In HeLa cells but not BHK cells the interaction of VSV-G and calnexin was prolonged in the presence of ionomycin. Taken together, the present results indicate that, by releasing Ca²⁺ from cellular storage, ionomycin inhibits ER-Golgi transport by interfering with the release of VSV-G from calnexin in HeLa cells. A mechanism of cell type-dependent ER-Golgi transport regulation was revealed. (J Oral Sci 57, 305-312, 2015)