Shiga toxin binds to globotriaosylceramide (Gb3) receptors on the target cell surface. To enter the cytosol, Shiga toxin is dependent on endocytic uptake, retrograde transport to the Golgi apparatus and further to the endoplasmic reticulum before translocation of the enzymatically active moiety to the cytosol. Here, we have investigated the importance of newly synthesized glycosphingolipids for the uptake and intracellular transport of Shiga toxin in HEp-2 cells. Inhibition of glycosphingolipid synthesis by treatment with either PDMP or Fumonisin B1 for 24–48 h strongly reduced the transport of Gb3-bound Shiga toxin from endosomes to the Golgi apparatus. This was associated with a change in localization of sorting nexins 1 and 2, and accompanied by a protection against the toxin. In contrast, there was no effect on transport or toxicity of the plant toxin ricin. High-resolution mass spectrometry revealed a 2-fold reduction in Gb3 at conditions giving a 10-fold inhibition of Shiga toxin transport to the Golgi. Furthermore, mass spectrometry showed that the treatment with PDMP (DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) and Fumonisin B1 among other changes of the lipidome, affected the relative content of the different glycosphingolipid species. The largest depletion was observed for the hexosylceramide species with the N-amidated fatty acid 16:0, whereas hexosylceramide species with 24:1 were less affected. Quantitative lipid profiling with mass spectrometry demonstrated that PDMP did not influence the content of sphingomyelins, phospholipids and plasmalogens. In contrast, Fumonisin B1 affected the amount and composition of sphingomyelin and glycolipids and altered the profiles of phospholipids and plasmalogens.