Overexpression of the DNA methyltransferase 3B (DNMT3B) gene and its effect on carcinogenesis has been demonstrated for various types of cancer. Recently, three single nucleotide polymorphisms (SNPs) of the DNMT3B promoter region, C46359T (-149C>T), -283T>C, and -579G>T have also been reported to be stratification markers that can predict an individual's susceptibility to cancers. In this study, we analyzed expression of DNMT3B in nasopharyngeal carcinoma (NPC) specimens and did not find elevated levels of DNMT3B in tumors using cDNA microarray analysis and RT-PCR. Meanwhile, 259 NPC patients and 250 controls were genotyped for the above three SNPs using a MALDI-TOF based mini-sequencing method. For C46359T (-149C>T), only the T/T genotype was found to be present in both patient and control groups (100% frequency). The frequency of the genotypes, -283CC, -283CT and -283TT, amongst NPC patients versus controls was, respectively, 86.1% versus 84.0%, 13.5% versus 15.6%, and 0.4% versus 0.4% (P=0.589). The allele frequency, -597TT, -597GT and -597GG, for patients versus controls was, respectively, 87.3% versus 84.8%, 12.0% versus 15.2%, and 0.8% versus 0 (P=0.501). The distribution of SNPs among cancer patients either featuring or not featuring cervical metastasis also did not reveal any significant difference. In conclusion, our data indicate that neither overexpression of DNMT3B nor the presence of three DNMT3B SNPs are associated with NPC, which suggests that DNMT3B might not play a role in hypermethylation of many tumor suppressor genes during carcinogenesis of NPC.