DNA methylation is crucial for normal development, but gene expression altered by DNA hypermethylation is often associated with human diseases, especially cancers. The gene TSPYL5, encoding testis-specific Y-like protein, was previously identified in microarray screens for genes induced by the inhibition of DNA methylation and histone deacetylation in glioma cell lines. The TSPYL5 showed a high frequency of DNA methylation-mediated silencing in both glioma cell lines and primary glial tumors. We now report that TSPYL5 is also inactivated by DNA methylation and could be a putative epigenetic target gene in gastric cancers. We found that the expression of TSPYL5 mRNA was frequently downregulated and inversely correlated with DNA methylation in seven out of nine gastric cancer cell lines. TSPYL5 mRNA expression was also restored after treating with a DNA methyltransferase inhibitor. In primary gastric tumors, methylation-specific PCR results in 23 of the 36 (63.9%) cases revealed that the hypermethylation at CpG islands of the TSPYL5 was detectable at a high frequency. Furthermore, TSPYL5 suppressed the growth of gastric cancer cells as demonstrated by a colony formation assay. Thus, strong associations between TSPYL5 expression and hypermethylation were observed, and aberrant methylation at a CpG island of TSPYL5 may play an important role in development of gastric cancers.