Carbenoxolone (CBX) is primarily used to relieve various types of neuropathic and inflammatory pain. However, little is known concerning the role of CBX in acute pain and its functional mechanisms therein and this was investigated in the present study. Rats underwent toe incision and behavioral tests were performed to assess mechanical hypersensitivity. The expression levels of pannexin 1 (Px1) and connexin 43 (Cx43) were detected using western blot analysis 2, 4, 6 or 24 h after toe incision, and the expression of TNF-α, IL-1β and P substance (SP) was determined by ELISA; Px1 and Cx43 expression was also examined by immunofluorescence staining. At 2, 6 and 12 h post-toe incision, the postoperative pain threshold was significantly reduced, which was subsequently recovered at 2 and 6 h post-surgery following pretreatment with CBX or pannexin 1 mimetic inhibitory peptide. CBX reduced Px1 levels at 4 and 24 h post-incision. However, Cx43 levels were reduced by CBX as little as 2 h post-surgery. Furthermore, CBX not only distinctly decreased the levels of Px1 and Cx43, but also reduced the co-localization of Px1 or Cx43 with glial fibrillary acidic protein, 2 h after incision. It was also observed that the protein levels of inflammatory makers (IL-1β, SP and TNF-α) showed a tendency to decline at 2, 4, 6 and 24 h after incision. Collectively, the expression of Px1 and Cx43 in astrocytes may be involved in pain behaviors diminished by CBX, and CBX potentially reduces acute pain by decreasing Px1 and Cx43 levels. Px1 and Cx43 from spinal astrocytes may serve important roles in the early stages and maintenance of acute pain, while preoperative injection of CBX has the potential to relieve hyperalgesia.