Protocadherins are a major subfamily of the cadherin superfamily, but little is known about their functions and intracellular signal transduction. We identified a homozygous loss of protocadherin 20 (PCDH20, 13q21.2) in the course of a program to screen a panel of non-small-cell lung cancer (NSCLC) cell lines (1 of 20 lines) for genomic copy number aberrations using an in-house array-based comparative genomic hybridization. PCDH20 mRNA was expressed in normal lung tissue but was not expressed in the majority of NSCLC cell lines without a homozygous deletion of this gene (10 of 19 lines, 52.6%). Expression of PCDH20 mRNA was restored in gene-silenced NSCLC cells after treatment with 5-aza 2'-deoxycytidine. The DNA methylation status of the PCDH20 CpG-rich region correlated inversely with the expression of the gene and a putative target region for methylation showed clear promoter activity in vitro. Methylation of this PCDH20 promoter was frequently observed in primary NSCLC tissues (32 of 59 tumors, 54.2%). Among our primary NSCLC cases, the methylated PCDH20 seemed to be associated with a shorter overall survival (P = 0.0140 and 0.0211 in all and stage I tumors, respectively; log-rank test), and a multivariate analysis showed that the PCDH20 methylation status was an independent prognosticator. Moreover, restoration of PCDH20 expression in NSCLC cells reduced cell numbers in colony formation and anchorage-independent assays. These results suggest that epigenetic silencing by hypermethylation of the CpG-rich promoter region of PCDH20 leads to loss of PCDH20 function, which may be a factor in the carcinogenesis of NSCLC.