When mice are treated with α-galactosylceramide (α-GalCer), NKT cells are activated and suppress the development of experimental airway inflammation. This suppressive effect is believed to be mediated by the upregulation of IFN-γ. Here, we investigated whether α-GalCer treatment can also modulate the development of experimental allergic conjunctivitis (EC). EC was induced in wild-type and IFN-γ-deficient Balb/c mice by active immunization with ragweed (RW) followed by challenge with RW in eye drops. The mice were intraperitoneally injected with α-GalCer or vehicle at the time of immunization or before RW challenge. Twenty-four hours after RW challenge, conjunctivas, spleens and sera were harvested for histological analysis, flow cytometric, proliferation and cytokine assays, and measurement of immunoglobulin levels, respectively. Treatment with α-GalCer at the time of the EC-priming immunization significantly increased Th2 responses and markedly upregulated the severity of the EC. However, treatment with α-GalCer just before the Ag challenge that triggers EC in primed animals significantly suppressed the disease. This was associated with an increased frequency of CD4 +CD25 + cells, which express Foxp3, in the spleen. α-GalCer treatment just prior to Ag challenge also suppressed the development of EC in IFN-γ-deficient mice, and we found apoptosis and anergy are unlikely to play a major role in the mechanism by which pre-challenge α-GalCer treatment suppresses EC. These data suggest that NKT cells can play a downregulatory role in the development of EC and that α-GalCer may be useful for treating allergic conjunctivitis.