MicroRNA-34 (miR-34), in particular miR-34a, has a negative regulatory effect on osteosarcoma cell proliferation, migration and invasion. Notably, it is also a post-transcriptional regulatory factor of (sex determining region Y)-box 2 (Sox-2), which is required for osteosarcoma cell self-renewal and tumorigenesis. As a direct regulator of Sox-2, miR-34a has been hypothesized to be greatly associated with the regulation of malignancies in osteosarcoma. To investigate the role of miR-34a in the malignancies of osteosarcoma, reverse transcription-quantitative polymerase chain reaction was performed to detect the expression level of miR-34a in osteospheres. The results revealed that the miR-34a, b and c were suppressed in osteosarcoma stem-like cells (OSCs) and osteospheres. The introduction of miR-34a mimics and short hairpin (sh) RNA targeting Sox-2 mRNA (shSox-2) in human OSCs markedly reduced their transformation properties in vitro and their capacity to form tumors in soft agar. Furthermore, the epigenetic expression of miR-34a and shSox-2 inhibited the expression of the stem cell marker, stem cell antigen-1 and led to the failure of osteosphere formation, respectively. The data of the present study indicated that the inhibitory role of miR-34a on tumor growth and metastasis of osteosarcoma may function by reducing the maintenance of osteosphere self-renewal capacity, elimination of tumorigenic ability and invasion of osteosarcoma in vitro. These findings may provide the basis for a novel therapeutic target of osteosarcomas based on inducing the expression of miR-34a.