To screen for methylation silenced genes in nasopharyngeal carcinoma cell line 5-8F, two-dimensional gel electrophoresis (2-DE) was performed to separate the proteins of treated and untreated 5-8F cells with demethylating agent 5-aza-2-dC, PDQuest software was used to analyze 2-DE images, and MALDI-TOF-MS was used to identify the differentially expressed proteins between the treated and untreated 5-8F cells. Then RT-PCR and Western blotting were performed to examine the expression levels of nm23-H1 mRNA and protein, one of the differential expression proteins, in the treated and untreated 5-8F cells, respectively. Methylation-specific PCR (MS-PCR) was performed to detect the methylated level of nm23-H1 gene in the treated and untreated 5-8F cells. 2-DE patterns of the treated and untreated 5-8F cells with 5-aza-2-dC were established, and a total of forty-nine differential protein spots were found in treated and untreated 5-8F cells. Thirty-three non-redundant differential proteins were identified by MS, 15 proteins of which were up-regulated after 5-aza-2-dC treatment. The results of Western blotting, RT-PCR and MS-PCR showed that nm23-H1 is a methylation silenced gene in 5-8F cell line. Encoding genes of 15 up-regulated proteins after 5-aza-2-dC treatment may be methylation silenced genes in NPC cell line 5-8F. The data will be helpful to screen for methylation silenced genes in nasopharyngeal carcinoma.