Simple SummaryBefore the union of an egg and spermatozoon, several vital processes occur for fertilization in the female reproductive system. One of these processes is the maturation of spermatozoa which occurs in the female reproductive tract. Spermatozoa not undergoing maturation in the female reproductive tract are unable to penetrate the egg. Many reports have suggested the involvement of different factors in mediating the functional maturation of spermatozoa. Follicular fluid (FF) is named as one of those factors. FF is an ovarian fluid that plays an essential role in egg maturation and sources extracellular vesicles (EVs). EVs are nano-containers that are released from different cells and are present in all body fluids. Several studies have reported that FF supports the functional maturation of spermatozoa. Therefore, we hypothesized that FF EVs might have a role in inducing functional maturation in spermatozoa. Surprisingly, the FF-derived EVs were able to aid vital functional parameters of spermatozoa and the effects from EVs were species- and source-specific. Therefore, deciphering the cargo of FF EVs responsible for modulating spermatozoa’s functions can potentially prove beneficial in diagnosing and treating male infertility and improving the current assisted reproductive technology protocols. While follicular fluid (FF) is known to enhance the functional properties of spermatozoa, the role of FF-derived extracellular vesicles (EVs) in this respect is unknown. We hypothesized that bovine FF EVs convey signals to spermatozoa supporting sperm viability, inducing sperm capacitation and acrosome reaction. In this study, the effects of bovine FF EVs on sperm functions are evaluated. Irrespective of the size of the follicles which FF EVs had originated from, they were capable of supporting sperm viability, inducing capacitation and acrosome reaction. These effects were specific to the source of bovine FF EVs, as human-cell-line-derived or porcine FF EVs did not affect spermatozoa viability or induced capacitation and acrosome reaction. A minimum of 5 × 105 EVs/mL was adequate to maintain sperm viability and induce capacitation and acrosome reaction in spermatozoa. Interestingly, with FF EV trypsin treatment, FF EVs lost their ability to support sperm functions. In conclusion, this study demonstrates that bovine FF EVs can support spermatozoa function and may contribute to a favorable periconceptional microenvironment. This is an important aspect of the interactions between different sexes at the earliest stages of reproduction and helps to understand molecular mechanisms modulating processes such as sperm competition and female cryptic choice.