Lateral and ventral lateral subregions of the periaqueductal gray (l/vlPAG) have been proved to be pivotal components in descending circuitry of itch processing, and their effects are related to the subclassification of neurons that were meditated. In the present study, lateral parabrachial nucleus (LPB), one of the most crucial relay stations in the ascending pathway, was taken as the input nucleus to examine the modulatory effect of l/vlPAG neurons that received LPB projections. Anatomical tracing, chemogenetic, optogenetic and local pharmacological approaches were utilized to investigate the participation of the LPB-l/vlPAG pathway in itch and pain sensation in mice. First, morphological evidence for projections from vesicular glutamate transporter-2 (VGluT2)-containing neurons in the LPB to l/vlPAG involved in itch transmission has been provided. Furthermore, chemogenetic and optogenetic activation of the LPB-l/vlPAG pathway resulted in both antipruritic effect and analgesic effect, whereas pharmacogenetic inhibition strengthened nociceptive perception without affecting spontaneous scratching behavior. Finally, in vivo pharmacology was combined with optogenetics which revealed that AMPA receptor-expressing neurons in l/vlPAG might play a more essential role in pathway modulation. These findings provide a novel insight about the connections between two prominent transmit nuclei, LPB and l/vlPAG, in both pruriceptive and nociceptive sensations, and deepen the understanding of l/vlPAG modulatory roles in itch sensation by chosen LPB as source of ascending efferent projections.Copyright © 2021 International Association for the Study of Pain.