It has been reported that costimulatory molecules, CD80/86–CD28 and CD154–CD40, critically contribute to activation of CD1d-restricted invariant NKT (iNKT) cells. Here we have demonstrated that ICOS, a new member of the CD28 family, plays a substantial role in iNKT cell activation. iNKT cells constitutively expressed ICOS as well as CD28 independently, and ICOS expression was further up-regulated 2–3 days after α-galactosylceramide (α-GalCer) treatment. Blockade of ICOS-mediated costimulation by administration of anti-ICOS ligand (B7RP-1) mAb or by ICOS gene knockout substantially inhibited α-GalCer-induced IFN-γ and IL-4 production, cytotoxic activity, and anti-metastatic effect. Moreover, blockade of both B7RP-1–ICOS and CD80/86–CD28 interactions mostly abolished the α-GalCer-induced immune responses. These findings indicate that iNKT cell activation is regulated by CD28 and IOCS independently.