Background & Aims: V alpha 14 invariant natural killer T cells (iNKT) are localized in peripheral tissues such as the liver rather than lymphoid tissues. Therefore, their role in modulating the stimulation of conventional, major histocompatibility complex (MHC)-restricted T-cell responses has remained ambiguous. We here describe a role for V alpha 14 iNKT cells in modulating conventional T-cell responses to antigen expressed in liver, using transferrin-moVA (Tf-mOVA) mice. Methods: Naive ovalbumin-specific class I MHC-restricted T cells (OTI) were adoptively transferred into Tf-mOVA mice in the presence or absence of iNKT-cell agonist alpha-galactosylceramide, after which OTI T-cell priming, antigen-specific cytokine production, cytotoxic killing ability, and liver damage were analyzed. Results: Transfer of OTI cells resulted in robust intrahepatic, antigen-specific proliferation of T cells. OTI T-cells were activated in liver, and antigen-specific effector function was stimulated by coactivation of V alpha 14 iNKT cells using alpha-galactosylceramide. This stimulation was absent in CD1d(-/-)Tf-mOVA mice, which lack Va14 iNKT cells, and was prevented when interferon-gamma and tumor necrosis factor-alpha production by V alpha 14 iNKT cells was blocked. Conclusions: CD1d-restricted V alpha 14 iNKT cells stimulate intrahepatic CD8 T-cell effector responses to antigen expressed in liver. Our findings elucidate a previously unknown intervention point for targeted immunotherapy to autoimmune and possibly infectious liver diseases.