The anti-neoplastic sphingolipid analog SH-BC-893 starves cancer cells to death by down-regulating cell surface nutrient transporters and blocking lysosomal trafficking events. However, the actual mechanism of action giving rise to these phenotypes remains unclear. Here, dynamic phosphoproteomics was used to further understand how the activity of PP2A is affected following cell treatment with SH-BC-893. These analyses combined with functional assays identified the differential regulation of Akt and Gsk3b by SH-BC-893 and C2-ceramide as responsible for the vacuolation of cells by SH-BC-893 but not C2-ceramide.