Cancer stem cells are a subpopulation of tumor cells with the ability of self-renewal and high differentiation potential and are thought to drive tumor initiation and growth. The resistance of cancer stem cells to therapy is the main problem in treatment, therefore intensive work is being done on the development of new therapies that would selectively target cancer stem cells, in which ascorbic acid has great potential. L-ascorbic acid is the only active form of vitamin C however, its low stability limits its therapeutic use. In order to increase chemical stability, its more stable lipophilic derivatives such as ascorbyl palmitate, can be packed into solid lipid nanoparticles and in that way delivered into cells. We examined the effect of ascorbate in free form and incorporated in solid lipid nanoparticles on tumor and healthy cells with a viability test and by measuring the intracellular level of reactive oxygen species. It was found that increased concentrations of ascorbate reduced cell viability and contribute to an increase in the level of reactive oxygen species, which indicates a pro-oxidative effect. We also examined the effect of the optimal source of ascorbate on the expression level of GAPDH protein and on the expression of osteosarcoma stem cell differentiation marker genes. We conclude that ascorbate affects cell metabolism by reducing the level of GAPDH protein, thus inhibiting the process of glycolysis, which causes energy crisis. Also, ascorbate maintains osteosarcoma stem cells at a lower level of differentiation by inducing increased expression of early differentiation markers genes while expression of late differentiation marker gene was not detected.