Triterpenoid saponins, synthesized via the mevalonic acid (MVA) pathway in the cytoplasm, provide protection against pathogens and pests in plants and health benefits for humans. However, the mechanisms how triterpenoid saponins are transported between cellular compartments remain uncharacterized. Here, we characterize a tonoplast localized multidrug and toxic compound extrusion transporter, GmMATE100 (encoded by Glyma.18G143700), from soybean (Glycine max L.). GmMATE100 is co-expressed with soyasaponin biosynthetic genes and its expression was induced by MeJA treatment, which also led to soyasaponin accumulation in soybean roots. GmMATE100 efficiently transports multiple type-B soyasaponins as well as type-A soyasaponins with low affinity from the cytosol to the vacuole in a yeast system. The GmMATE100 loss-of-function mutant showed a significant decrease in type A and type-B soyasaponin contents in soybean roots. This study not only characterized the first soybean triterpenoid saponin transporter, but also provided new knowledge for the rational engineering soyasaponin content and composition in soybean plants to modulate their levels within the crop environments.