Natural killer T (NKT) cells are a newly identified T-cell population with potential immunomodulatory functions. Several studies have shown modulating effects of NKT cells activated by α-galactosylceramide, a model antigen, on NK cell function. We here report a differential modulating effect of NKT cells on the interferon-γ (IFN-γ) production and cytolytic function of NK cells in a chlamydial infection model, using NKT-cell-deficient mice and antibody blocking (anti-CD1d monoclonal antibody) approaches. Our results showed that both NKT and NK cells became activated and produced IFN-γ following Chlamydia muridarum infection in vitro and in vivo. The NK cells in NKT-cell-deficient mice and CD1d-blocked mice showed decreased CD69 expression, cellular expansion and IFN-γ production but surprisingly showed increased cytolytic activity (degranulation) of immature and more mature NK cell subsets, suggesting an inhibitory role of NKT cells on NK cell killing activity. The results suggest that NKT cells preferentially promote IFN-γ production but are inhibitory for the cytotoxic function of NK cells in this infection model. Furthermore, the differential modulating effect of NKT cells on the IFN-γ production and cytotoxicity of NK cells was observed in immature and mature NK cell subsets, although it was more dramatic in the relatively mature CD11b(high)  CD27(high) NK cell subset. This finding demonstrates the complexity of innate cell interactions in infection and the possible differential impact of NKT cells on the variable functional aspects of other cell(s) even in one infection setting