A vacuolar H+-ATPase (VHA) gene (ZMVHA-B1) was isolated from an eelgrass (Zostera marina) leaf cDNA library and was characterized to be approximately 1.4 kbp in length and to encode the B subunit protein of VHA comprising 488 amino acids. ZMVHA-B1 was highly expressed in all organs of eelgrass; the expression level was highest in the leaves. On transformation of a yeast vma2 null mutant with ZMVHA-B1, yeast cells became able to grow at pH 7.5, accompanied by the vesicular accumulation of LysoSensor green DND-189. Thus, ZMVHA-B1 expressed in yeast cells produced a functional B subunit that was efficiently incorporated into the VHA complex and eventually restored vacuolar morphology and activity. This success expedites the application of heterologous expression in yeast mutant cells to the screening of eelgrass genes involved in salt-resistance mechanisms, which are to be utilized in improving important crops.