MicroRNA (miR)-21 has been found to be overexpressed in osteosarcoma (OS). The aim of the present study was to investigate the effect of miR-21-5p on the Warburg effect and stemness maintenance in OS cells and its potential molecular mechanism. Herein, miR-21-5p was overexpressed or inhibited in MG-63 cells via transfection with mimics or inhibitors. The effect of miR-21-5p on cell viability, apoptosis, Warburg effect and stemness maintenance were explored in OS cells. The results demonstrated that miR-21-5p inhibition suppressed MG-63 cell viability and enhanced their apoptosis. Additionally, miR-21-5p inhibition attenuated the stemness maintenance of MG-63 cells, as demonstrated by the reduced proportion of CD133-positive MG-63 cells, the decrease in tumorsphere formation capacity, and the downregulation of Sox2, Oct4, and Nanog proteins. Moreover, miR-21-5p inhibition suppressed the Warburg effect in MG-63 cells, as indicated by the decrease in glucose uptake, lactic acid production, and ATP level and the downregulation of proteins involved in the Warburg effect (GLUT1, LDHA, HK2, and PKM2). Furthermore, the results suggested that the effect of miR-21-5p suppression on stemness and the Warburg effect may be associated with the decreased activity of the Wnt/β-catenin pathway in OS cells. Our findings suggest a novel potential biomarker for OS therapy.