An important aspect of tumor progression is the ability of cancer cells to escape detection and clearance by the immune system. Recent studies suggest that several tumors express soluble factors interfering with the immune response. Here, we show that semaphorin-3A (Sema-3A), a secreted member of the semaphorin family involved in axonal guidance, organogenesis, and angiogenesis, is highly expressed in several tumor cells. Conditioned media of Sema-3A-transfected COS-7 cells or human recombinant Sema-3A inhibited primary human T-cell proliferation and cytokines production under anti-CD3 plus anti-CD28 stimulating conditions. Sema-3A also inhibited the activation of nonspecific cytotoxic activity in mixed lymphocyte culture (MLC), as measured against K-562 cells. In contrast, suppression of Sema-3A in tumor cells with a small interfering RNA (siRNA) augmented T-cell activation. The inhibitory effect of Sema-3A in T cells is mediated by blockade of Ras/mitogen-activated protein kinase (MAPK) signaling pathway. The presence of Sema-3A increased the activation of the Ras family small GTPase Rap1 and introduction of the dominant-negative mutant of Rap1 (Rap1N17) blunted the immunoinhibitory effects of Sema-3A. These results suggest that Sema-3A inhibits primary T-cell activation and imply that it can contribute to the T-cell dysfunction in the tumor microenvironment.