Osteosarcoma (OS) is the most common type ofbone cancer, with a peak incidence in the early childhood.Emerging evidence suggests that treatments targeting cancerstem cells (CSCs) within a tumor can halt cancer and improvepatient survival. MicroRNAs (miRNAs) have been implicatedin the maintenance of the CSC phenotype, thus, identificationof CSC-related miRNAs would provide information for abetter understanding of CSCs. Downregulation of miRNA-29family members (miR-29a/b/c; miR‑29s) was observed inhuman OS, however, little is known about the functions ofmiR-29s in human OS CSCs. Previously, during the characterizationof 3AB-OS cells, a CSC line selected from humanOS MG63 cells, we showed a potent downregulation ofmiR-29b. In this study, after stable transfection of 3AB-OScells with miR-29b-1, we investigated the role of miR-29b-1in regulating cell proliferation, sarcosphere-forming ability,clonogenic growth, chemosensitivity, migration and invasiveability of 3AB-OS cells, in vitro. We found that, miR-29b-1overexpression consistently reduced both, 3AB-OS CSCsgrowth in two- and three-dimensional culture systems andtheir sarcosphere- and colony-forming ability. In addition, while miR-29b-1 overexpression sensitized 3AB-OS cells tochemotherapeutic drug-induced apoptosis, it did not influencetheir migratory and invasive capacities, thus suggesting acontext-depending role of miR-29b-1. Using publicly availabledatabases, we proceeded to identify potential miR-29btarget genes, known to play a role in the above reportedfunctions. Among these targets we analyzed CD133, N-Myc,CCND2, E2F1 and E2F2, Bcl-2 and IAP-2. We also analyzedthe most important stemness markers as Oct3/4, Sox2 andNanog. Real-time RT-PCR and western-blot analyses showedthat miR-29b-1 negatively regulated the expression of thesemarkers. Overall, the results show that miR-29b-1 suppressesstemness properties of 3AB-OS CSCs and suggest that developingmiR-29b-1 as a novel therapeutic agent might offerbenefits for OS treatment.