Sphingomyelinase (SMase) from Bacillus cereus (Bc-SMase) hydrolyzes sphingomyelin (SM) to phospho-choline and ceramide in a divalent metal ion-dependent manner, and is a virulence factor for septicemia. Bc-SMase has three characteristic sites, viz., the central site (catalytic site), side-edge site (membrane binding site), and beta-hairpin region (membrane binding site). Here, we show that the beta-hairpin directly binds to gangliosides, especially NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1ceramide (GM3) through a carbohydrate moiety. Neuraminidase inhibited the binding of Bc-SMase to mouse peritoneal macrophages in a dose-dependent manner. SPR analysis revealed that the binding response of Bc-SMase to liposomes containing GM3 was about 15-fold higher than that to liposomes lacking GM3. Moreover, experiments with sitedirected mutants indicated that Trp-284 and Phe-285 in the beta-hairpin play an important role in the interaction with GM3. The binding of W284A and F285A mutant enzymes to mouse macrophages decreased markedly in comparison to the binding by wild-type enzymes. Therefore, we conclude that GM3 is the primary cellular receptor for Bc-SMase, and that the beta-hairpin region is the tethering region for gangliosides. Crown Copyright (C) 2013 Published by Elsevier Inc. All rights reserved.