Brain orexin/hypocretin (orx/hcrt) cells regulate energy balance and sleep-wake cycles. Glucose can inhibits orx/hcrt cells, but whether other dietary macronutrients have similar effects is unclear. We found that orx/hcrt cells are stimulated by nutritionally-relevant mixtures of amino acids (AAs). This was seen in both in brain slice patch-clamp experiments, and in c-Fos expression assays following central or peripheral administration of AAs to mice in vivo. Physiological mixtures of AAs activated orx/hcrt cells through a dual mechanism involving inhibition of KATP channels and activation of system-A amino acid transporters. Non-essential AAs were more effective than essential AAs. Moreover, physiological concentrations of AAs suppressed the inhibitory glucose responses of orx/hcrt cells. Contrary to previous models, these results imply that orx/hcrt cells sense macronutrient balance, rather than net energy value, in extracellular fluid.