β-Mannosylceramide (b-ManCer) is an iNKT cell agonist that induces protection against tumors through a TNF-α-NOS dependent pathway, distinct from the IFN-Υ-dependent pathway induced by a-GalCer and other α-linked ligands.. One enigma remaining is how this agonist with β-linked sugar moiety is presented by CD1d.  In this study, we asked whether monoclonal antibodies, L363 and L317, specific for an α-GalCer-CD1d complex, could bind to a β-ManCer-CD1d complex.  Surprisingly both antibodies recognized β-ManCer-CD1d complexes.  The antibodies also inhibited activation of both iNKT cell hybridomas and primary splenic iNKT cells.  The magnitude of inhibition of β-ManCer-induced stimulation by L363 was greater than that of α-GalCer.  In contrast, L363 did not bind to a sulfatide-CD1d complex or inhibit activation of a type II NKT cell hybridoma.  Distinct from the iNKT cell TCR that can reorient the glycosyl head of glycolipids protruding from  the CD1d groove to assume a structure like that of α-GalCer-CD1d, L363 has been reported not to be able to reorient the glycosyl head.  Thus, these results suggest that the β-ManCer-CD1d complex has a three-dimensional structure similar to that of the α-GalCer-CD1d complex despite its beta-linked sugar moiety.