Here we show that depletion of pyruvate, non-essential amino acid and glucose increases the effects of metformin on MDA-MB-231 cells. The effects of metformin differ also between monolayer culture and tumour spheroids. Our results indicate that in glucose-depleted media metformin supressed MDA-MB-231 cell proliferation and reduced their survival due to inhibition of oxidative phosphorylation. In the absence of glucose, MDA-MB-231 cells were unable to adapt to energy crises induced by metformin via upregulation of glycolysis. Thus, metformin probably supressed cellular proliferation in an AMPK-independent manner. Since the absence of glucose increases the effects of metformin on MDA-MB-231 cells, metformin could target cancer cells in a poorly perfused core of the tumour. Medium renewal blocked the anti-proliferative effects of metformin in the presence of 5,6 mM of glucose, which might explain inconsistent results in previous studies. Metformin reduces the number of MDA-MB-231 cells synergistically with 2-DG, which is a competitive inhibitor of glycolysis. Finally, we have observed that combined treatment with metformin and 2-DG induces detachment of viable MDA-MB-231 and MCF-7 cells from the bottom of cell culture wells, which might be important to better understand the effects of changes in metabolic pathways on the process of cancer cell metastazation.