Semaphorin 3B (SEMA3B) is a secreted member of the semaphorin family, important in axonal guidance. We and others have shown that SEMA3B can act as a tumor suppressor by inducing apoptosis either by reexpression in tumor cells or applied as a soluble ligand. The common method of inactivation of SEMA3B is by allele loss and tumor-acquired promoter methylation. We studied the mechanism of SEMA3B-induced tumor cell apoptosis and found that vascular endothelial growth factor (VEGF)₁₆₅ significantly decreased the proapoptotic and antimitotic effect of transfected or secreted SEMA3B on lung and breast cancer cells. VEGF₁₆₅ binds to neuropilin, receptors for SEMA3B, and we found that SEMA3B competed for binding of ¹²⁵I-VEGF₁₆₅ to lung and breast cancer cells. We also found that small interfering RNA knockdown of tumor-produced VEGF-A or the use of an anti-VEGF neutralizing antibody (Ab) significantly inhibited tumor cell growth in vitro. By contrast, VEGF₁₂₁, a VEGF variant that lacks binding to neuropilin (NP)-1 or NP-2 receptors, was not expressed in tumor cells and had no effect on SEMA3B growth-suppressing activities. In conclusion, we hypothesize that VEGF₁₆₅, produced by tumor cells, acts as an autocrine survival factor and that SEMA3B mediates its tumor-suppressing effects, at least in part, by blocking this VEGF autocrine activity.