Lung cancer in never smokers (NS) shows striking demographic, clinicopathological and molecular distinctions from the disease in smokers (S). Studies on selected genetic and epigenetic alterations in lung cancer identified that the frequency and profile of some abnormalities significantly differ by smoking status. This study compared the transcriptome of lung adenocarcinoma cell lines derived from S (n= 3) and NS (n= 3) each treated with vehicle (control), histone deacetylation inhibitor (trichostatin A) or DNA methylation inhibitor (5-aza-2′-deoxycytidine). Among 122 genes reexpressed following 5-aza-2′-deoxycytidine but not trichostatin A treatment in two or more cell lines (including 32 genes in S-only and 12 NS-only), methylation was validated for 80% (98/122 genes). After methylation analysis of 20 normal tissue samples and 14 additional non–small cell lung cancer cell lines (total 20), 39 genes frequently methylated in normal (>20%, 4/20) and 21 genes rarely methylated in non–small cell lung cancer (≤10%, 2/20) were excluded. The prevalence for methylation of the remaining 38 genes in lung adenocarcinomas from S (n= 97) and NS (n= 75) ranged from 8–89% and significantly differs between S and NS forCPEB1,CST6,EMILIN2,LAYNandMARVELD3(P< 0.05). Furthermore, methylation ofEMILIN2,ROBO3andIGDCC4was more prevalent in advanced (Stage II–IV,n= 61) than early (Stage I,n= 110) tumors. Knockdown ofMARVELD3, one of the novel epigenetically silenced genes, by small interfering RNA significantly reduced anchorage-independent growth of lung cancer cells (P< 0.001). Collectively, this study has identified multiple, novel, epigenetically silenced genes in lung cancer and provides invaluable resources for the development of diagnostic and prognostic biomarkers.