Carbon ion radiotherapy (CIRT) is more effective than conventional photon beam radiotherapy in treating osteosarcoma (OSA); however, the outcomes of CIRT alone are still unsatisfactory. In this study, we aimed to investigate whether miR-29b acts as a radiosensitizer for CIRT. The OSA cell lines U2OS and KHOS were treated with carbon ion beam alone, γ-ray irradiation alone, or in combination with an miR-29b mimic. OSA cell death as well as invasive and migratory abilities were analyzed through viability, colony formation, Transwell, and apoptosis assays. miR-29 expression was downregulated in OSA tissues compared to that in normal tissues and was associated with metastasis and relapse in patients with OSA. Further, miR-29b was found to directly target the transcription factor Sp1 and suppress the activation of the phosphatase and tensin homolog (PTEN)-AKT pathway. Conversely, Sp1 was found to attenuate the inhibitory effects of miR-29b in OSA cells. When used in combination with miR-29b mimic, carbon ion beam markedly inhibited invasion, migration, and proliferation of OSA cells and promoted apoptosis by inhibiting AKT phosphorylation in a Sp1/PTEN-mediated manner. Taken together, miR-29b mimic improved the radiosensitivity of OSA cells via the PTEN-AKT-Sp1 signaling pathway, presenting a novel strategy for the development of carbon ion beam combination therapy.