Ovarian granulosa cells are essential to gonadotrophin-regulated estrogen production, female cycle maintenance and fertility. The epithelial Na channel (ENaC) is associated with female fertility; however, whether and how it plays a role in ovarian cell function(s) remained unexplored. Here, we report patch-clamp and Na imaging detection of ENaC expression and channel activity in both human and mouse ovarian granulosa cells, which are promoted by pituitary gonadotrophins, follicle stimulating hormone (FSH) or luteinizing hormone (LH). Cre-recombinase- and CRISPR-Cas9-based granulosa-specific knockout of ENaC α subunit (Scnn1a) in mice resulted in failed estrogen elevation at early estrus, reduced number of corpus luteum, abnormally extended estrus phase, reduced litter size and subfertility in adult female mice. Further analysis using technologies including RNA sequencing and Ca imaging revealed that pharmacological inhibition, shRNA-based knockdown or the knockout of ENaC diminished spontaneous or stimulated Ca oscillations, lowered the capacity of intracellular Ca stores and impaired FSH/LH-stimulated transcriptome changes for estrogen production in mouse and/or human granulosa cells. Together, these results have revealed a previously undefined role of ENaC in modulating gonadotrophin signaling in granulosa cells for estrogen homeostasis and thus female fertility.