CD1d-restricted natural killer T (iNKT) cells have been shown to provide adjuvant activity against cancer by producing interferon (IFN)-γ. However, the role of invariant NKT (iNKT) cells in the tumor microenvironment has not yet been fully addressed. Our aim is to elucidate the antitumor effect of iNKT cells in the tumor microenvironment by using an intrathoracic murine malignant pleural mesothelioma model that we had previously developed and to provide pleural effusion as a good surrogate of the tumor microenvironment. We found that the number of iNKT cells increased dramatically in the pleural effusion after intrathoracic tumor cell injection at an earlier phase compared with accumulation of CD8 T cells. These iNKT cells showed increased expression of CD25 and increased ratio of cells positive for IFN-γ intracellular staining. iNKT cells sorted from pleural effusion of tumor burden mice produced larger amount of IFN-γ compared with naive mice. Mice pretreated in vivo with anti-CD1d-blocking Ab showed increased amount of pleural effusion and decreased ratio of total and effector-type CD8 T cells as well as decreased intracellular IFN-γ expression of CD8T-cell in the pleural effusion. In vivo administration of α-galactosylceramide (α-GalCer) showed prolonged survival associated with less pleural effusion and increased ratio of IFN-γ-positive iNKT cells and CD8 T cells in the pleural effusion. Therefore, this study suggests that iNKT cells accumulating in the tumor microenvironment play an antitumor effect by producing IFN-γ and enhancing subsequent CD8 T-cell response. Furthermore, in vivo administration of α-GalCer could suppress mesothelioma growth by activating iNKT cells.