The etiology of neuropathic pain is mostly caused by mechanical deformation and neuroinflammation, of which neuroinflammation is the main cause of chronic neuropathic pain. Activation of the TLR4/NF-κB signaling pathway mediates elevated levels of inflammatory cytokines, and we clearly demonstrated by in vivo and in vitro Western blot experiments that β-sitosterol significantly inhibited the elevated Toll-like receptor 4 (TLR4) expression levels and nuclear factor-kappa B (NF-κB) activation associated with inflammatory responses. In cellular experiments, we clearly saw that both β-sitosterol and TLR4/NF-κB signaling pathway inhibitors could inhibit M1 proinflammatory phenotype expression and promote M2 anti-inflammatory phenotype expression in GMI-R1 microglia by flow cytometry and immunofluorescence assays. Therefore, we suggest that β-sitosterol can affect microglial polarization by inhibiting the TLR4/NF-κB signaling pathway thereby reducing neuroinflammation and thus alleviating neuropathic pain.© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.