B7-H3 is a B7-family co-stimulatory molecule and is broadly expressed on various tissues and immune cells. Transduction of B7-H3 into some tumours enhances anti-tumour responses. We have recently found that a triggering receptor expressed on myeloid cell-like transcript 2 (TLT-2) is a receptor for B7-H3. Here, we examined the roles of tumour-associated B7-H3 and the involvement of TLT-2 in anti-tumour immunity. Ovalbumin (OVA)(257-264)-specific OT-I CD8(+) T cells exhibited higher cytotoxicity against B7-H3-transduced OVA-expressing tumour cells (B7-H3/E.G7) in vitro and selectively eliminated B7-H3/E.G7 cells in vivo. The presence of B7-H3 on target cells efficiently augmented CD8(+) T-cell-mediated cytotoxicity against alloantigen or OVA, whereas the presence of B7-H3 in the priming phase did not affect the induced cytotoxicity. B7-H3 transduction into five tumour cell lines efficiently reduced their tumorigenicity and regressed growth. Treatment with either anti-B7-H3 or anti-TLT-2 monoclonal antibody accelerated growth of a tumour that expressed endogenous B7-H3, suggesting a co-stimulatory role of the B7-H3-TLT-2 pathway. The TLT-2 was preferentially expressed on CD8(+) T cells in regional lymph nodes, but was down-regulated in tumour-infiltrating CD8(+) T cells. Transduction of TLT-2 into OT-I CD8(+) T cells enhanced antigen-specific cytotoxicity against both parental and B7-H3-transduced tumour cells. Our results suggest that tumour-associated B7-H3 directly augments CD8(+) T-cell effector function, possibly by ligation of TLT-2 on tumour-infiltrating CD8(+) T cells at the local tumour site.