The homopentameric B-subunit of bacterial protein Shiga toxin (STxB) binds to the glycolipid Gb(3) in plasma membranes, which is the initial step for entering cells by a clathrin-independent mechanism. It has been suggested that protein clustering and lipid reorganization determine toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb(3) clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb(3) lipid structure was investigated by means of high resolution methods such as fluorescence and scanning force microscopy. STxB was found to form protein clusters on homogenous 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol/Gb(3) (65:30:5) bilayers. In contrast, membranes composed of DOPC/cholesterol/sphingomyelin/Gb(3) (40:35:20:5) phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb(3), STxB-Gb(3) complexes organize within the liquid ordered phase upon protein binding. Our findings suggest that STxB is capable of forming a new membrane phase that is characterized by lipid compaction. The significance of this finding is discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations.