Abstract Vacuolar H +-ATPases (V-ATPases) are ATP-dependent proton pumps localized at membranes of intracellular acidic organelles and plasma membranes of various cell types. By virtue of its regulation in acidification, V-ATPase is required for many intracellular processes such as receptor-mediated endocytosis and protein sorting. Here we report the molecular characterization of the E subunit of V-ATPase in Caenorhabditis elegans. This subunit is one of the most well conserved subunits sharing approximately 57% identity with the human homologue, ATP6E. Green fluorescent protein (GFP) and whole-mount immunostaining analyses showed that V-ATPase E subunit ( vha-8) is abundantly expressed in the H-shaped excretory cell, consistent with the expression patterns observed for other V-ATPase subunits. Double-stranded RNAs (or RNAi) targeted to vha-8 resulted in embryonic and larval lethality for the first filial generation, indicating that vha-8 is essential during early developmental processes. In addition, accumulation of abnormal endomitotic oocytes and defects in receptor-mediated endocytosis were observed in parental animals. These findings suggest that multiple phenotypes caused by the disruption of pH homeostasis are due to the defective V-ATPase. In summary, vha-8 encoding the E subunit of V-ATPase in C. elegans is essential for embryogenesis and receptor-mediated endocytosis.