Mesenchymal stem cell-derived extracellular vesicle (MSC-EV) has shown promise for pain relief, but its efficacy is limited. Preconditioning MSC with tumor necrosis factor-α (TNF-α) may enhance their therapeutic potential; however, the impact on analgesia and underlying mechanisms remains unclear. Here, we investigated the analgesic effects of EV from TNF-α-preconditioned MSC (T-EV) in a chronic constriction injury (CCI) mouse model and examined the molecular mechanisms involved. Following intrathecal injection, T-EV produced greater improvements in mechanical and thermal pain thresholds than control MSC-EV (C-EV), achieving enhanced pain relief for two weeks. Whole-cell patch-clamp recordings revealed that T-EV markedly decreased both the firing rate and action potential amplitude of dorsal root ganglion (DRG) neurons. RNA sequencing revealed that T-EV was enriched in miR-101b-3p. Silencing miR-101b-3p in T-EV abolished their enhanced analgesic effects and reversed DRG hyperexcitability. Moreover, miR-101b-3p was shown by luciferase assays to bind directly to the 3'UTR of Nav1.6, suppressing its expression. Engineered MSC-derived nanovesicle overexpressing miR-101b-3p replicated the increased pain relief observed with T-EV. These findings demonstrate that TNF-α preconditioning improves the analgesic potency of MSC-EV by delivering miR-101b-3p, which downregulates Nav1.6 and decreases DRG hyperexcitability. This study supports the therapeutic potential of miR-101b-3p-enriched vesicle as a novel strategy for treating neuropathic pain.