The chlamydial glycolipid exoantigen (GLXA), a glycolipid antigen derived from Chlamydia muridarum, has been implicated in chlamydial-host cell interaction. Although glycolipid antigens from Sphingomonas and related bacteria have been shown to activate invariant natural killer T (iNKT) cells, it is not yet known whether GLXA can activate these cells. In this study, we have for the first time investigated the role of GLXA in iNKT cell activation using in vitro as well as in vivo settings. First, we examined the effect of GLXA on iNKT cell activation in a cell-free antigen-presentation assay, and found that GLXA specifically stimulated iNKT1.4 hybridoma cell produce enhanced amounts of IL-2. Next, we analyzed the effect of pharmacological activation of iNKT cells by GLXA using iNKT cell-deficient (iNKT knockout (KO)) mice and bone marrow-derived dendritic cell (BMDC)-liver mononuclear cell (LMC) coculture system. On stimulation with GLXA, iNKT cells produced higher quantities of cytokines in a CD1d-dependent fashion. More importantly, iNKT cells from GLXA-treated, but not from cell mock-treated, mice showed higher expression of activation marker, CD69, and enhanced production of interferon (IFN)-γ and IL-4 in vivo. Cumulatively, these data provide evidence on the pharmacological ability of GLXA in specifically activating iNKT cells