Endometrial epithelium exhibits a robust ion transport activity required for dynamical regulation of uterine fluid environment and thus embryo implantation. However, there still lacks a thorough understanding of the ion transport processes and regulatory mechanism in peri-implantation endometrial epithelium. As a gaseous signaling molecule or gasotransmitter, hydrogen sulfide (H 2 S) regulates a myriad of cellular and physiological processes in various tissues, including the modulation of ion transport proteins in epithelium. This study aimed to investigate the effects of H 2 S on ion transport across mouse endometrial epithelium and its possible role in embryo implantation. The existence of endogenous H 2 S in pregnant mouse uterus was tested by the detection of two key H 2 S-generating enzymes and measurement of H 2 S production rate in tissue homogenates. Transepithelial ion transport processes were electrophysiologically assessed in Ussing chambers on early pregnant mouse endometrial epithelial layers, demonstrating that H 2 S suppressed the anion secretion by blocking cystic fibrosis transmembrane conductance regulator (CFTR). H 2 S increased intracellular Cl− concentration ([Cl−] i) in mouse endometrial epithelial cells, which was abolished by pretreatment with the CFTR selective inhibitor CFTR inh -172. The cAMP level in mouse endometrial epithelial cells was not affected by H 2 S, indicating that H 2 S blocked CFTR in a cAMP-independent way. In vivo study showed that interference with H 2 S synthesis impaired embryo implantation. In conclusion, our study demonstrated that H 2 S inhibits the transepithelial anion secretion of early pregnant mouse endometrial epithelium via blockade of CFTR, contributing to the preparation for embryo implantation. • The expression and activity of CBS/CTH exhibited an increasing tendency in mouse uterus during early pregnancy. • H 2 S decreased I SC in pregnant mouse endometrial epithelium via inhibition of CFTR. • H 2 S increased [Cl−] i of mouse endometrial epithelial cells by blockade of CFTR. • Interference with H 2 S synthesis in vivo partially impaired embryo implantation. [ABSTRACT FROM AUTHOR]