Down-regulated expression of human leukocyte antigen (HLA) class I molecules in many human cancers facilitate tumor cells to escape from immune attack. Promoter hypermethylation, one of the major epigenetic changes responsible for gene inactivation, plays an important role in gastric carcinogenesis. This study evaluated the expression and alteration of HLA class I molecules in a panel of 47 pairs of gastric cancer specimens with their noncancerous parts from Chinese patients by using immunohistochemistry (IHC), reverse transcription polymerase chain reaction (RT-PCR) and methylation-specific PCR (MSP) analysis. The expression of HLA-A, HLA-B/C and HLA class I complex was lost or down-regulated in human gastric cancer. The percentage of promoter methylation was 59.57% for HLA-A gene, 55.32% for HLA-B gene and 48.94% for HLA-C gene in gastric cancer, while it was decreased to 19.15%, 12.77% and 6.38% in the adjacent nontumor tissues, respectively. Seven of 10 (70%), 4 of 6 (66.7%) and 3 of 4 (75%) gastric cancer specimens with promoter hypermethylation at HLA-A, -B and -C loci showed transcriptional inactivation of HLA-A,-B and -C genes, suggesting an association between promoter hypermethylation and down-regulated expression of HLA class I molecules. Human gastric cancer cell line BGC-823 showed HLA-A down-regulation with promoter methylation of HLA-A locus. Treatment with DNA methyltransferase inhibitor restored the expression of HLA-A mRNA and surface HLA-A complex. Thus, our results showed that promoter hypermethylation might be one of the mechanisms that lead to HLA class I antigen down-regulation in gastric cancer.