To investigate the roles of lactotransferrin gene (LTF, also referred to as the lactoferrin gene, LF), located at 3p21.3 within the common minimal deletion region, in the pathogenesis of nasopharyngeal carcinoma (NPC), we first detected its expression level in 33 primary NPC tissues and 15 chronic nasopharyngitis tissues. Absent expression or downregulation of LTF were observed in 76% (25 of 33) of primary NPC tissues. We further found that 25% (5 of 20) of NPC specimens had loss of heterozygosity (LOH) at the LTF locus. LTF mutation assessed by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing was noted in 30% (6 of 20) of primary NPC tissues. In addition, hyper-methylation of LTF promoter region was found in 63.6% (21 of 33) of primary NPC samples but not in chronic nasopharyngitis tissues. The LTF transcripts in NPC cell lines increased upon treatment with the demethylation compound, 5-aza-2-deoxycytidine. In conclusion, our data indicate that two-hit silencing of LTF through genetic and epigenetic changes may be a common and important event in the carcinogenesis of NPC.