Neuropathic pain is a complex condition that seriously affects human quality of life. This study aimed to investigate the therapeutic mechanism of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) and try to discover new targets for alleviating neuropathic pain. Extracellular vesicles were isolated and identified via ultracentrifugation. BV-2 microglial cells were stimulated with lipopolysaccharide (LPS) in the presence or absence of MSC-EVs. Further, microglial activation and neuroinflammation were evaluated by flow cytometry, RT-qPCR, and ELISA. High-throughput sequencing analysis was performed to reveal the differentially expressed (DE) miRNAs in BV-2 microglia. Autophagy-related regulators were assessed by Western blotting and Immunofluorescence staining. Chronic constriction injury (CCI) model was used to induce neuropathic pain in rats, and the mechanical withdrawal threshold (MWT) was measured. High-throughput sequencing analysis identified 17 DE miRNAs, which were mainly enriched in PI3K-AKT and mTOR signaling pathways. MSC-EVs inhibited the activation of PI3K/AKT/mTOR signaling pathway in LPS-stimulated microglia. Moreover, MSC-EVs treatment enhanced the autophagy level in activated microglia, whereas autophagy inhibitor 3-MA reversed the suppressing effects of MSC-EVs on microglial activation and neuroinflammation. The MSC-EV-mediated transfer of miR-99b-3p was verified to promote microglial autophagy, and miR-99b-3p overexpression suppressed the expression of pro-inflammatory factors in activated microglia. During in vivo studies, intrathecal injection of MSC-EVs significantly up-regulated the expression of miR-99b-3p, and alleviated mechanical allodynia caused by activated microglia in the spinal cord dorsal horn of CCI rats. Moreover, MSC-EVs treatment repaired CCI-induced autophagic impairment by stimulating autophagy in the spinal cord. Collectively, our findings demonstrated that MSC-EVs had an analgesic effect on neuropathic pain via promoting autophagy, and these antinociceptive effects were at least in part caused by MSC-EV-mediated transfer of miR-99b-3p, thereby inhibiting microglial activation and pro-inflammatory cytokines expression.