<p>Itch is a common symptom among patients suffering dermatological and systemic diseases, yet effective clinical treatments are currently lacking. Previous research has suggested that vesicular glutamate transporter 3 (VGLUT3)-lineage sensory neurons may play a role in inhibiting itch, but the circuit mechanisms within the spinal cord remain unclear. In this study, we employed optogenetic techniques to activate VGLUT3-lineage sensory afferents in mice and observed a significant reduction in scratching behaviors elicited by both pruritogens and mechanical stimuli. Moreover, aversive component of chemical itch assessed by conditioned place aversion (CPA) was abrogated. Viral tracing combined with electrophysiological recordings revealed synaptic connections between VGLUT3⁺ sensory neurons and spinal dynorphin (SC^DYN) /neuropeptide Y-expressing (SC^NPY) neurons. Further pharmacological studies indicated that intrathecal injection of antagonists of neuropeptide Y1 receptor and kappa opioid receptor (KOR) separately diminished VGLUT3⁺ neurons-mediated inhibitory effects on mechanical and chemical itch, respectively. In summary, our findings suggest that VGLUT3⁺ sensory neurons participate in itch regulation through interactions with two classes of inhibitory neurons in the spinal cord, shedding light on potential therapeutic targets for distinct forms of itch management.</p>