Hyperoside, a natural flavonoid, exhibits a wide range of biological activities, including analgesic effects on acute and chronic inflammatory pain. This study illustrates that repeated intraperitoneal administration or microinjection of hyperoside into the ventrolateral periaqueductal grey (vlPAG) alleviated mechanical allodynia, cold allodynia, and abnormal gait induced by spared nerve injury (SNI) in male mice. Furthermore, repeated hyperoside administration suppressed SNI-induced astrocyte reactivity in the vlPAG. Moreover, hyperoside alleviated the pain behaviors resulting from the pharmacogenetic activation of vlPAG astrocytes. These results suggest that hyperoside may effectively mitigate neuropathic pain and inhibit astroglial reactivity in the vlPAG, highlighting its potential as a viable therapeutic intervention for chronic neuropathic pain.