Extracellular vesicles (EVs) are membrane-bound vesicles containing various proteins, lipids, and nucleic acids. EVs are found in many body fluids, such as blood and urine. The release of EVs can facilitate intercellular communication through fusion with the plasma membrane or endocytosis into the recipient cell or through internalization of the contents. Recent studies have reported that EVs isolated from human endometrial epithelial cells (EECs) promote sperm fertilization ability. EVs from uterine flushing fluid more closely resemble the physiological condition of the uterus. However, it is unclear whether EVs derived directly from uterine flushing fluid have the same effect on sperm. This study aimed to research the effect of EVs from uterine flushing fluid on sperm.EVs were isolated from the uterine flushing fluid. The presence of EVs was confirmed by nanoparticle tracking analysis (NTA), Western blot, and transmission electron microscopy (TEM). EVs were incubated with human sperm for 2 h and 4 h. The effects of EVs on sperm were evaluated by analyzing acrosome reaction, sperm motility, and reactive oxygen species (ROS).The EVs fractions isolated from the uterine fluid were observed in cup-shaped vesicles of different sizes by TEM. All isolated vesicles contained similar numbers of vesicles in the expected size range (30-200 nm) by NTA. CD9 and CD63 were detected in EVs by western blot. Comparing the motility of the two groups incubated sperm motility significantly differed at 4 h. The acrosome reactions were promoted by incubating with EVs significantly. ROS were increased in sperm incubated with EVs.Our results showed EVs present in the uterine fluid. Acrosome reactions and ROS levels increased in human sperm incubated with EVs. EVs from uterine fluid can promote the capacitation of human sperm. The increased capacitation after sperm interaction with EVs suggests a possible physiological effect during the transit of the uterus.©2024 Deng et al.