Osteosarcoma (OS) is a malignant bone cancer with severe chromosomal abnormalities and genetic aberrations. Our previous work reported the dedifferentiation of OS, which is related to poor prognosis. However, the molecular mechanism that regulates OS dedifferentiation is still a subject of exploration. Emerging evidence has suggested that microRNAs (miRNAs) are associated with the pathogenesis of OS and could potentially be developed for use as diagnostic biomarkers and therapeutic strategies. In the present study, we intended to illustrate the role of miR-34a in the dedifferentiation of OS. Upregulation of miR-34a was observed while OS cells were induced into stem-like phenotype. Notably, inhibition of miR-34a could promote the reprogramming transition of OS. Further exploration on the downstream network of miR-34a identified that blocking plasminogen activator inhibitor-1 (PAI-1) expression could restrain OS dedifferentiation into cancer stem-like cells by downregulating SRY-related-HMG box (Sox) 2. We also showed that Sox2 overexpression rescued the suppression phenotype driven by PAI-1 inhibition. Conversely, PAI-1 inhibitor (PAI-039) could suppress the upregulation of Sox2 expression caused by miR-34a inhibition. Be applying bone extracellular matrix (BEM)-OS models, we demonstrated the phenotypic heterogeneity of OS cells, consistent with a strong concordance between PAI-1 and Sox2 expression levels. Taken together, our findings proved miR-34a to be a bona fide suppressor involved in the regulation of OS dedifferentiation. Targeting miR-34a or its direct target PAI-1 could offer new strategies for OS treatment.