We have here studied the role of cholesterol in transport of ricin from endosomes to the Golgi apparatus. Ricin is endocytosed even when cells are depleted for cholesterol by using methyl-β-cyclodextrin (mβCD). However, as here shown, the intracellular transport of ricin from endosomes to the Golgi apparatus, measured by quantifying sulfation of a modified ricin molecule, is strongly inhibited when the cholesterol content of the cell is reduced. On the other hand, increasing the level of cholesterol by treating cells with mβCD saturated with cholesterol (mβCD/chol) reduced the intracellular transport of ricin to the Golgi apparatus even more strongly. The intracellular transport routes affected include both Rab9-independent and Rab9-dependent pathways to the Golgi apparatus, since both sulfation of ricin after induced expression of mutant Rab9 (mRab9) to inhibit late endosome to Golgi transport and sulfation of a modified mannose 6-phosphate receptor (M6PR) were inhibited after removal or addition of cholesterol. Furthermore, the structure of the Golgi apparatus was affected by increased levels of cholesterol, as visualized by pronounced vesiculation and formation of smaller stacks. Thus, our results indicate that transport of ricin from endosomes to the Golgi apparatus is influenced by the cholesterol content of the cell.