Cystic ovarian follicles result in reproductively inefficient dairy cows. Fibroblast growth factor-9 (FGF9) gene expression in granulosa cells (GC) is downregulated in cystic vs. non-cystic follicles in cattle. FGF9 is thought to act as an intraovarian regulator by stimulating proliferation and reducing steroid production by GC and theca cells (TC). Analysis using Affymetirx GeneChip bovine Genome Arrays (n = 8) revealed that microRNA-221 (MIR221) in TC was upregulated by FGF9. We selected this gene for further study due to recent evidence indicating MIR221 is involved in cell proliferation, angiogenesis and cancer. We hypothesized that MIR221 is regulated during follicle growth and by steroids and hormones that induce cAMP. Three experiments were conducted to evaluate hormonal regulation of MIR221 mRNA in TC and its possible role in follicular development using sensitive real-time PCR (U6 as housekeeping gene). TC were isolated from large (>8 mm) follicles (n = 6 to 8 per pool) collected from an abattoir, cultured for 48 h in medium with 10% fetal calf serum, and then treated in serum-free medium for an additional 24 h. Data were analyzed via GLM procedure of SAS. Exp. 1 tested effects of cAMP pharmacologic agents (dbcAMP at 0.1 mg/mL; forskolin at 4.1 μg/mL) on FGF9-induced MIR221 mRNA abundance; neither affected (P > 0.10) MIR221 expression whereas FGF9 increased (P < 0.05) MIR221 abundance 1.94-fold. Exp. 2 tested the effect of 300 ng/mL of steroids (estradiol; androstenedione) and phytoestrogens (genestein; α- and β-zearanol) on MIR221; none of the treatments influenced (P > 0.10) MIR221 mRNA levels (3.45 ± 0.62 relative abundance). In Exp. 3, follicular fluid (FF) was collected from ovaries obtained at an abattoir, and MIR221 mRNA abundance measured; MIR221 mRNA was 3-fold greater (P < 0.05) in FF aspirated from small (1 to 5 mm) follicles compared with FF from large follicles. These novel results indicate MIR221 is increased by FGF9 but not regulated by steroids or cAMP. Greater levels of MIR221 in FF of small vs. large follicles suggest a role for FGF9-induced MIR221 in follicular development.