Regulation of estradiol production, central to ovarian follicular development and reproductive function, is mediated by a complex interaction of pituitary gonadotropins such as FSH with locally produced regulatory molecules. We have previously demonstrated a negative association of expression of Cocaine-and Amphetamine-Regulated Transcript (CART) with follicle health status and a novel local negative role for CART in regulation of basal estradiol production by bovine granulosa cells. However, effects of CART on FSH-induced estradiol production and the underlying mechanism(s) mediating the physiological actions of CART on granulosa cells are not known. Objectives of the present study were to determine effects of CART on basal and FSH-induced intracellular cAMP levels, aromatase mRNA, estradiol accumulation, calcium signaling and the intracellular signaling pathways involved using primary cultures of bovine granulosa cells. CART treatment potently inhibits the FSH-induced rise in granulosa cell cAMP levels, estradiol accumulation and aromatase mRNA. Furthermore, results show that calcium is essential for FSH-induced cAMP and estradiol accumulation and CART significantly inhibits FSH-induced calcium influx. Select G-protein and protein kinase inhibitors were used to elucidate pathways involved in CART actions. The inhibitory actions of CART on FSH signaling and estradiol production are mediated via a G_o/i-dependent pathway, while none of the other signaling inhibitors had any effect on CART actions. Results demonstrate novel potent inhibitory effects of CART on multiple components of the FSH signaling pathway linked to estradiol production and follicular development and shed new insight into the mechanism of action of CART potentially pertinent within and beyond the reproductive system.