Some synthetic and bacterial glycolipids presented by CD1d specifically activate invariant NKT (iNKT) cells bearing an invariant Vα14-Jα18 (mouse) or Vα24-Jα18 (human) TCR. The antigenic glycolipids identified to date consist of two hydrophobic chains and an α-glycoside in which the 2'-OH group is in the cis orientation toward the anomeric group, namely, either an α-galactoside or an α-glucoside. Several microbial α-mannosyl glycolipids, in which the 2'-OH group is in the trans orientation, were herein examined to establish whether they have potential to activate iNKT cells. We found that α-mannnosyl1-3 (6'-O-acyl α-mannosyl)-1-1 monoacylglycerol and cholesteryl 6'-O-acyl α-mannoside, found in Saccharopolyspora and Candida albicans, respectively, induced the activation of iNKT cells, dependent on CD1d. In contrast, α-mannosyldiacylglycerol found in Streptococcus suis or α-mannosylceramide demonstrated markedly less antigenicity for iNKT cells. The potentially antigenic α-mannosyl glycolipids contributed to the protection of mice against infection with S. pneumoniae in which iNKT cells have previously been found to participate. Furthermore, these glycolipids induced the production of proinflammatory cytokines by macrophages, thereby suggesting their recognition by specific pattern recognition receptors (PRRs). Collectively, these results suggest that these microbial α-mannosyl glycolipids are capable of being recognized by both the invariant TCR and PRRs and inducing immune responses.