Neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus (ARC) play a central role in stimulation of feeding. They sense and integrate peripheral and central signals, including ghrelin and leptin. However, the mechanisms of interaction of these hormones in NPY neurons are largely unknown. This study explored the interaction and underlying signaling crosstalk between ghrelin and leptin in NPY neurons. Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in single neurons isolated from ARC of adult rats was measured by fura-2 microfluorometry. Ghrelin increased [Ca²⁺]_i in 31% of ARC neurons. The [Ca²⁺]_i increases were inhibited by blockers of phospholipase C, adenylate cyclase, and protein kinase A (PKA). Ghrelin-induced [Ca²⁺]_i increases were suppressed by subsequent administration of leptin. Fifteen of 18 (83%) of ghrelin-activated, leptin-suppressed neurons contained NPY. Leptin suppression of ghrelin responses was prevented by pretreatment with inhibitors of phosphatidylinositol 3-kinase (PI3K) and phosphodiesterase 3 (PDE3), but not mitogen-activated protein kinase. ATP-sensitive potassium channel inhibitors and activators did not prevent and mimic leptin suppression, respectively. Though leptin phosphorylated signal-transducer and activator of transcription 3 (STAT3) in NPY neurons, neither STAT3 inhibitor nor genetic STAT3 deletion altered leptin suppression of ghrelin responses. Furthermore, orexigenic effect of intracerebroventricular ghrelin in rats was counteracted by leptin in a PDE3-dependent manner. These findings indicate that ghrelin increases [Ca²⁺]_i via mechanisms depending on phospholipase C and adenylate cyclase-PKA pathways in ARC NPY neurons, and that leptin counteracts ghrelin responses via PI3K-PDE3 pathway. This interaction may play an important role in regulating ARC NPY neuron activity and, thereby, feeding.