Invariant natural killer T (iNKT) cells are crucial for host defense against a variety of microbial pathogens, but the underlying mechanisms of iNKT cells activation by microbes are not fully explained. In this study, we investigated the molecular mechanisms of iNKT cell activation in lipopolysaccharide (LPS)-stimulated preterm birth using an adoptive transfer system and diverse neutralizing antibodies (Abs) and inhibitors. We found that adoptive transfer of decidual iNKT cells to LPS-stimulated iNKT cell deficient Jα18−/− mice that lack invariant Vα14Jα281T cell receptor (TCR) expression significantly decreased the time to delivery and increased the percentage of decidual iNKT cells. Neutralizing Abs against Toll-like receptor 4 (TLR-4), CD1d, interleukin (IL)-12 and IL-18, and inhibitors blocking the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated kinase (ERK) significantly reduced in vivo percentages of decidual iNKT cells, their intracellular interferon (IFN)-γ production and surface CD69 expression. In vitro, in the presence of the same Abs and inhibitors used as in vivo, decidual iNKT cells co-cultured with LPS-pulsed dendritic cells (DCs) showed significantly decreased extracellular and intracellular IFN-γ secretion and surface CD69 expression. Our data demonstrate that the activation of decidual iNKT cells plays an important role in inflammation-induced preterm birth. Activation of decidual iNKT cells also requires TLR4-mediated NF-κB, MAPK p38 and ERK pathways, the proinflammatory cytokines IL-12 and IL-18, and endogenous glycolipid antigens presented by CD1d.