The increasing evidence supported the role of Enhancer of Zeste Homolog 2 (EZH2) in the cancer development and progression. However, its precise role in the tumorigenesis of Nasopharyngeal Carcinoma (NPC) remains to be elucidated. EZH2 was depleted by retroviral infection in the NPC cells (HK-1, CNE-2, CNE-1 and C666-1). The degree of EZH2 knockdown was then assessed by real-time quantitative PCR and Western Blot analysis. Cell proliferation was assessed using the soluble tetrazolium salt (MTS) cell proliferation assay, and cell cycle was measured by FACS test. The methylation status of p16(INK4a) was determined by bisulphate treatment of the DNA, followed by MSP. EZH2 was over-expressed in NPC cells, and the expression in undifferentiated-derived NPC cells (CNE-1, C666-1) was more significant than differentiated-derived NPC cells (HK-1, CNE-2). EZH2 was successfully depleted after retroviral infection in C666-1 cells, and the EZH2 depletion could inhibit the proliferation and arrested G1/S phase of NPC cells. In addition, both mRNA and protein levels of p16(INK4a) increased significantly in presence of EZH2 depletion. The further Methylation-Specific Polymerase Chain Reaction (MSP) assay suggested that over-expressed EZH2 may contribute to the reduction of p16(INK4a) expression by hyper methylating its promoter. EZH2 is overexpressed in NPC and reduces expression of p16(INK4a) by influencing methylation, opening therapeutic options.