Aerolysin of the Gram‐negative bacterium Aeromonas hydrophila consists of small (SL) and large (LL) lobes. The α‐toxin of Gram‐positive Clostridium septicum has a single lobe homologous to LL. These toxins bind to glycosylphosphatidylinositol (GPI)‐anchored proteins and generate pores in the cell's plasma membrane. We isolated CHO cells resistant to aerolysin, with the aim of obtaining GPI biosynthesis mutants. One mutant unexpectedly expressed GPI‐anchored proteins, but nevertheless bound aerolysin poorly and was 10‐fold less sensitive than wild‐type cells. A cDNA of N‐acetylglucosamine transferase I (GnTI) restored the binding of aerolysin to this mutant. Therefore, N‐glycan is involved in the binding. Removal of mannoses by α‐mannosidase II was important for the binding of aerolysin. In contrast, the α‐toxin killed GnTI‐deficient and wild‐type CHO cells equally, indicating that its binding to GPI‐anchored proteins is independent of N‐glycan. Because SL bound to wild‐type but not to GnTI‐deficient cells, and because a hybrid toxin consisting of SL and the α‐toxin killed wild‐type cells 10‐fold more efficiently than GnTI‐deficient cells, SL with its binding site for N‐glycan contributes to the high binding affinity of aerolysin.