Gb3 (Gal α1-4, Gal β1-4, Glc β1-1, Ceramide) is a neutral glycosphingolipid specifically expressed in several human tissues, and is recognised as a receptor for Shiga toxins. Gb3 has been demonstrated to be upregulated in a number of cancer types , which makes it a feasible target for cancer diagnosis and therapy. However, little is known about the regulation of Gb3 biosynthesis in the cells, and further investigations are needed. In this study, a quantitative real time RT-PCR technique was used to investigate the changes in Gb3 synthase gene (Gb3syn) expression in several cancer cell lines. We demonstrated that Gb3syn expression was upregulated in response to higher cell density and longer culturing time in HEp-2 cells, while no regulation was detected in other cell lines tested. The effects of two Gb3 synthesis modulating compounds, 2-deoxy-D-glucose (2DG) and sodium butyrate, were also studied. 2DG inhibited the expression of Gb3syn in HEp-2 cells and induced cell protection against Shiga toxin (Stx) and diphtheria toxin (DT). The binding and endocytosis of Stx was not affected by 2DG, and cell sensitivity to Stx was not rescued by pyruvate indicating that the protection observed after 2DG treatment was not due to lack of ATP. Thus it was concluded that later steps of intoxication by Stx, retrograde transport to the Golgi apparatus and the endoplasmic reticulum and/or translocation to the cytosol, were perturbed by 2DG-treatment and led to cell protection against the toxin. In contrast, sodium butyrate upregulated Gb3syn expression in colon cancer cells, and increased their sensitivity to Stx. The data indicated that two distinct mechanisms, changed intracellular transport and/or higher binding of the toxins, were responsible for cell sensitisation by sodium butyrate. Finally, the relation between Gb3 and cancer cell metastatic potential was studied using a live time-lapse imaging combined with immunofluorescence microscopy, which allowed us to analyse individual cells. The preliminary data suggest that there is a correlation between Gb3 exposed on the plasma membrane and the motility of cancer cells.