--- - |2+ Abstract Aim Serotonin (5‐hydroxytryptamine, 5‐HT), an important neurotransmitter and hormone, modulates many physiological functions including body temperature. We investigated neural mechanisms involved in the inhibition of brown adipose tissue (BAT) sympathetic nerve activity (SNA) and BAT thermogenesis evoked by 5‐HT. Methods Electrophysiological recordings, intravenous (iv) injections and nanoinjections in the brains of anesthetized rats. Results Cooling‐evoked increases in BAT SNA were inhibited by the intra‐rostral raphé pallidus (rRPa) and the iv administration of the 5‐HT1A receptor agonist, 8‐OH‐DPAT, or 5‐HT. The intra‐rRPa 5‐HT, the intra‐rRPa and the iv 8‐OH‐DPAT, but not the iv 5‐HT‐induced inhibition of BAT SNA were prevented by nanoinjection of a 5‐HT1A receptor antagonist in the rRPa. The increase in BAT SNA evoked by nanoinjection of NMDA in the rRPa was not inhibited by iv 5‐HT, indicating that iv 5‐HT does not inhibit BAT SNA by acting in the rRPa or in the sympathetic pathway distal to the rRPa. In contrast, under a warm condition, blockade of 5HT1A receptors in the rRPa increased BAT SNA and BAT thermogenesis, suggesting that endogenous 5‐HT in the rRPa contributes to the suppression of BAT SNA and BAT thermogenesis. The increases in BAT SNA and BAT thermogenesis evoked by nanoinjection of NMDA in the dorsomedial hypothalamus (DMH) were inhibited by iv 5‐HT, but those following bicuculline nanoinjection in the DMH were not inhibited. Conclusions The systemic 5‐HT‐induced inhibition of BAT SNA requires a GABAergic inhibition of BAT sympathoexcitatory neurons in the DMH. In addition, during warming 5‐HT released endogenously in rRPa inhibits BAT SNA. - 'Acta Physiologica, Volume 0, Issue ja, -Not available-. '