The present study was attempted to investigate the effect of puerarin, an isoflavone compound isolated from Pueraria lobata, on both the basal body temperature and pyrogenic fever in unanesthetized, restrained rats. Intraperitoneal administration of puerarin or crude extracts of Pueraria lobata elicited hypothermia. Direct administration of a small amount of puerarin into the lateral cerebral ventricle produced the same extent of hypothermia. Systemic or central administration of puerarin causes a decrease in both colonic temperature and hypothalamic 5-HT efflux in rats. The puerarin-induced hypothermia and decreased 5-HT efflux in the hypothalamus were attenuated by selective depletion of hypothalamic 5-HT produced by intracerebroventricular injection of 5,7-dihydroxytryptamine. Furthermore, the puerarin-induced hypothermia was almost completely abolished by treatment with a 5-HT_2A-receptor agonist (DOI or quipazine) or a 5-HT_1A-receptor antagonist [(−)-pindolol]. A 5-HT_2A-receptor antagonist (ketanserin) or a 5-HT_1A-receptor agonist (8-OH-DPAT) had additive effects with puerarin. Intracerebroventricular administration of interleukin-1 caused an increase in both colonic temperature and hypothalamic 5-HT efflux. The interleukin-1-induced hyperthermia and increased 5-HT efflux in the hypothalamus were attenuated by treatment with systemic administration of puerarin. The data indicate that puerarin exerts its hypothermic and antipyretic effects by activating 5-HT_1A receptor and/or antagonizing 5-HT_2A receptors in the hypothalamus.