Objective: To characterize well-represented microRNAs in human follicular fluid (FF) and to ascertain whether they are cargo of FF exosomes and whether they are involved in the regulation of follicle maturation. Design: FF exosomes were characterized by nanosight, flow cytometry, and exosome-specific surface markers. Expression microRNA profiles from total and exosomal FF were compared with those from plasma of the same women. Setting: University laboratory and an IVF center. Patient(s): Fifteen healthy women who had undergone intracytoplasmic sperm injection. Intervention(s): None. Main Outcome Measure(s): TaqMan low-density array to investigate the expression profile of 384 microRNAs; DataAssist and geNorm for endogenous control identification; significance analysis of microarrays to identify differentially expressed microRNAs; nanosight, flow-cytometry, and bioanalyzer for exosome characterization; bioinformatic tools for microRNAs target prediction, gene ontology, and pathway analysis. Result(s): We identified 37 microRNAs upregulated in FF as compared with plasma from the same women. Thirty-two were carried by microvesicles that showed the well-characterized exosomal markers CD63 and CD81. These FF microRNAs are involved in critically important pathways for follicle growth and oocyte maturation. Specifically, nine of them target and negatively regulate mRNAs expressed in the follicular microenvironment encoding inhibitors of follicle maturation and meiosis resumption. Conclusion(s): This study identified a series of exosomal microRNAs that are highly represented in human FF and are involved in follicular maturation. They could represent noninvasive biomarkers of oocyte quality in assisted reproductive technology.