Aim Spinal neuroinflammation contributes to the mechanism of stress-induced hyperalgesia (SIH). Recent research has demonstrated that bone marrow mesenchymal stem cells (BMSCs) alleviate chronic pain. However, what remains unidentified is whether BMSCs could improve hyperalgesia induced by chronic restraint stress (CRS). In another dimension, our previous study proved that gut microbiota played an important role in CRS-induced hyperalgesia in mice. Yet, whether BMSCs treatments change gut microbiota composition in CRS mice remains unexplored.Mechanical allodynia and thermal hyperalgesia were used to assess pain behavior. Composition of fecal samples were verified by 16S rRNA analysis. Western blot was used to investigate the expression of adenosine monophosphate-activated protein kinase (AMPK)/ nuclear factor kappa B (NF-κB) signaling pathway, pro-inflammatory cytokines [interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), IL-6], and the markers of microglia and astrocytes. The morphology of glia cells was evaluated by immunofluorescence staining.CRS down-regulated phosphorylated AMPK (p-AMPK), up-regulated phosphorylated NF-κB p65 (p-NF-κB p65), activated microglia and astrocytes and promoted the secretion of IL-1β, TNF-α and IL-6 in the spinal cord. BMSCs alleviated CRS-induced hyperalgesia by inhibiting the activation of microglia and astrocytes and by reducing neuroinflammation via improving the disrupted AMPK/NF-κB pathway. Furthermore, BMSCs also raised the relative abundance of Muribaculaceae and Lachnospiraceae in CRS mice feces, which was significantly related to its effect of relieving hyperalgesia.Our results support that BMSCs could alleviate CRS-induced hyperalgesia by reducing AMPK/NF-κB-dependent neuroinflammation in the spinal cord and restoring the homeostasis of gut microbiota.Copyright © 2022. Published by Elsevier Inc.