Forkhead L2 (Foxl2) is expressed in ovarian granulosa cells and participates in steroidogenesis by transcriptionally regulating target genes such as steroidogenic acute regulatory protein (StAR) and CYP19A1. In this study, a direct link between microRNA-133b (miR-133b) and Foxl2-mediated estradiol release in granulosa cells was established. miR-133b was involved in follicle-stimulating hormone (FSH)-induced estrogen production. Luciferase assays confirmed that miR-133b was bound to the 3' untranslated region (3'UTR) of Foxl2 mRNA. Consistent with this finding, miR-133b overexpression reduced the Foxl2 levels. Furthermore, miR-133b inhibited Foxl2 binding to the StAR and CYP19A1 promoter sequences. These results demonstrate that miR-133b down-regulates Foxl2 expression in granulosa cells by directly targeting the 3'UTR, thus inhibiting the Foxl2-mediated transcriptional repression of StAR and CYP19A1to promote estradiol production.