Propofol is the most widely used intravenous general anesthetic; however, the neuronal circuits that mediate its anesthetic effects are still poorly understood. Glutamatergic neurons in the lateral hypothalamus have been reported to be involved in maintenance of arousal and consciousness. Using Vglut2-Cre transgenic mice, we recorded this group of cells specifically and found that propofol can directly inhibit the glutamatergic neurons, and enhance inhibitory synaptic inputs on these cells, thereby reducing neuronal excitability. Through chemogenetic interventions, we found that inhibition of these neurons increased the duration of propofol-induced anesthesia and reduced movement in the animals after the recovery of right reflex. In contrast, activating this group of cells reduced the duration of propofol anesthesia and increased the animals' locomotor activity after the recovery of right reflex. These results suggest that propofol-induced anesthesia involves the inhibition of glutamatergic neurons in the lateral hypothalamus.