Cancer-induced bone pain (CIBP) represents the pain induced by bone metastases from malignancies. The role of extracellular vesicles (Evs) has been underscored in bone metastasis. However, the function of Evs, especially these derived from M2 macrophages (M2φ-Evs) in CIBP is unclear. Therefore, this investigation aimed to probe the possible antinociceptive effect of M2φ-Evs in CIBP and the underlying mechanism of action. Using the C57bl/6 mice, a CIBP animal model was established by the administration of Walker 256 mammary gland carcinoma cells, followed by M2φ-Evs administration. It was found that CIBP mice treated with M2φ-Evs had significantly reduced nociception and serum inflammatory factors. Microarray sequencing revealed that microRNA-216a (miR-216a) was the most upregulated miRNA in Evs-treated mouse spinal cord tissues. Subsequent bioinformatics, GSEA and KEGG enrichment analyses demonstrated that HMGB1 and TLR4-NF-κB pathway were the downstream effectors of miR-216a and were both downregulated in spinal cord tissues of CIBP mice treated with M2f-Evs. Rescue experiments displayed that after we reduced miR-216a expression in M2φ-Evs, the antinociceptive effect of M2φ-Evs on CIBP mice was inhibited, and the HMGB1 expression and the TLR4-NF-κB signaling were significantly activated. Together, M2φ-Evs relieve CIBP by carrying miR-216a, which was elicited through the HMGB1/TLR4-NF-κB axis. © 2021 E-Century Publishing Corporation. All rights reserved.