Small noncoding RNA molecules (miRNA) regulate protein levels in a post-transcriptional manner by partial base pairing to the 3'-UTR of target genes thus mediating degradation or translational repression. Previous studies indicate that numerous miRNA regulate the biosynthesis of intraovarian hormones, and emerging evidence indicates that one of these, miRNA-221 (MIR221), may be a modulator of ovarian function. However, the hormonal control of ovarian MIR221 is not known. The objectives of this study were to investigate the developmental and hormonal regulation of MIR221 expression in granulosa (GC) and theca cell (TC) and its possible role in regulating follicular function. Bovine ovaries were collected from a local abattoir and GC and TC were obtained from small (<6 mm) and large (≥8 mm) follicles. In Exp. 1, GCs of small follicles had 9.7-fold greater (P < 0.001) levels of MIR221 than those of large follicles, and TCs of large follicles had 3.7-fold greater (P < 0.001) levels of MIR221 than those of small follicles. In large follicles, abundance of MIR221 was 66.6-fold greater (P < 0.001) in TCs than in GCs. In small follicles, MIR221 abundance did not differ (P = 0.14) between GC and TCs. In vitro Exp. 2, 3, and 4 revealed that treatment of bovine TCs with various steroids, phytoestrogens, IGF1, forskolin, and dibutyryl cyclic adenosine monophosphate had no effect (P > 0.35) on MIR221 expression, whereas treatment with fibroblast growth factor 9 (FGF9) and FGF2 increased (P < 0.001) TC MIR221 abundance 1.7- to 2.5-fold. In Exp. 5, FGF9 increased (P < 0.05) GC MIR221 abundance by 1.7- and 2.0-fold in small and large follicles, respectively. The role of MIR221 in GC steroidogenesis was investigated in Exp. 6 and it was found that transfection with a MIR221 mimic reduced (P < 0.01) GC estradiol and progesterone production induced by FSH and IGF1, whereas transfection with MIR221 inhibitor had little or no effect. We conclude that thecal MIR221 expression is increased by FGF9 and increased MIR221 may act to inhibit GC steroidogenesis in cattle.