Angiogenesis is essential for the growth and maturation of the ovarian follicle and its transition into the corpus luteum. In addition to the main proangiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), follicular fluid (FF) contains the hormone prolactin (PRL), which is known to promote angiogenesis in vivo. Here, we show that FF from large follicles, which contains twice the PRL level of FF from small follicles, stimulates endothelial cell proliferation to a greater extent than the latter, and that immunoneutralization of PRL prevents FF from stimulating endothelial cell proliferation. Notably, the FF increases the expression of the short and long PRL receptor isoforms in endothelial cells, and a purified PRL standard stimulates endothelial cell proliferation but only after the cells have been pretreated with FF. However, purified PRL activates the JAK2/STAT3 pathway in endothelial cells in the absence of pretreatment with FF. In summary, PRL present in the FF stimulates the proliferation of endothelial cells. This effect likely involves the upregulation of the short and long PRL receptor isoforms and is independent of PRL-induced JAK2/STAT3 signaling.