Neuropeptide Y (NPY) is a potent feeding stimulant. The orexigenic effect of NPY might be caused in part by the action of Y1 receptors. However, the existence of multiple NPY receptors including a possible novel feeding receptor has made it difficult to determine the relative importance of the Y1 receptor in feeding regulation. Herein we certified that the Y1 receptor is a major feeding receptor of NPY by using the potent and selective Y1 antagonist (−)-2-[1-(3-chloro-5-isopropyloxycarbonylaminophenyl)ethylamino]-6-[2-(5-ethyl-4-methyl-1,3-thiazol-2-yl)ethyl]-4-morpholinopyridine (J-115814) and Y1 receptor-deficient (Y1−/−) mice. J-115814 displaced¹²⁵I-peptide YY binding to cell membranes expressing cloned human, rat, and murine Y₁ receptors withK _i values of 1.4, 1.8, and 1.9 nM, respectively, and inhibited NPY (10 nM)-induced increases in intracellular calcium levels via human Y1 receptors (IC₅₀ = 6.8 nM). In contrast, J-115814 showed low affinities for human Y2 (K _i > 10 μM), Y4 (K _i = 640 nM) and Y5 receptors (K _i = 6000 nM). Intracerebroventricular (ICV) (10–100 μg) and intravenous (IV) (0.3–30 mg/kg) administration of J-115814 significantly and dose-dependently suppressed feeding induced by ICV NPY (5 μg) in satiated Sprague-Dawley rats. Intraperitoneal (IP) administration of J-115814 (3–30 mg/kg) significantly attenuated spontaneous feeding in db/db and C57BL6 mice. Feeding induced by ICV NPY (5 μg) was unaffected by IP-injected J-115814 (30 mg/kg) in Y1−/− mice and was suppressed in wild-type and Y5−/− mice. These findings clearly suggest that J-115814 inhibits feeding behaviors through the inhibition of the typical Y1 receptor. We conclude that the Y1 receptor plays a key role in regulating food intake.