The aim of this study was to evaluate the promoter methylation status and loss of heterozygosity (LOH) of the SEMA3B in non-small cell lung cancers (NSCLCs). We analyzed the methylation status of semaphorin 3B (SEMA3B) promoter and LOH at 3p21.3 in eight NSCLC cell lines and 27 primary tumors. Hypermethylation of SEMA3B was found in 50% of the cell lines and 41% of the primary tumors studied. The presence of hypermethylation was statistically associated with loss of SEMA3B expression in both cell lines (P = 0.02) and primary tumors (P < 0.01). There was no correlation between SEMA3B and tumor stage. On the other hand, the correlation between SEMA3B methylation status and LOH at 3p21.3 was significant (P = 0.02). Notably, 7 of 8 tumors with both hypermethylation and LOH of SEMA3B showed the absence of the expression. Treatment with 5-AZAC restored SEMA3B expression in NSCLC cell line. These results indicate that SEMA3B gene alterations may play a important role in the malignant transformation of NSCLC via a two-hit mechanism, including epigenetic changes and allelic loss, for tumor suppressor gene inactivation.