Background <p>Osteosarcoma (OS) is the most common non-hematogenous primary malignancy in the bone. Due to several origins of OS, 30–40% OS patients would experience recurrence and metastasis, with a 5-year survival rate of 20–30%. Mesenchymal stem cells (MSCs) transform into OS cells during the differentiation into osteoblasts, and circular RNA (circRNA) hsa_circ_0003611 might contribute to the differentiation of MSCs into osteoblasts. However, the role of hsa_circ_0003611 in the transformation of MSCs into OS cells is largely unknown. This study aims to investigate whether hsa_circ_0003611 tunes the transformation of MSCs into OS cells.</p> Methods <p>Here, human bone marrow mesenchymal stem cells (hBMSCs) with hsa_circ_0003611 stably silenced was constructed. Moreover, protein-RNA interaction was detected by RNA immunoprecipitation (RIP), and N⁶-methyladenosine (m⁶A) modification of hsa_circ_0003611 was determined using methylated RNA immunoprecipitation (MeRIP).</p> Results <p>The present study reveals that hsa_circ_0003611 level is almost absent in OS cells compared to that in osteoblasts and MSCs. Moreover, hsa_circ_0003611 silence enhances the transformation of MSCs into OS cells in vitro and triggered tumorigenicity of MSCs for OS in vivo. Mechanistically, silence of hsa_circ_0003611 promotes the transformation of MSCs into OS cells by activating MYC proto-oncogene, bHLH transcription factor (MYC) via insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3). Moreover, hsa_circ_0003611 silence improves MYC mRNA stability by facilitating the association between IGF2BP3 and MYC mRNA in MSCs. Furthermore, m⁶A modification disrupts the association between hsa_circ_0003611 and IGF2BP3 to enhance the association between IGF2BP3 and MYC mRNA in MSCs.</p> Conclusion <p>In summary, these findings highlight the role of hsa_circ_0003611 in the transformation of MSCs into OS cells and provide novel targets and strategies for OS treatment.</p>