Nasopharyngeal carcinoma (NPC) is a commonly occurring tumor in southern China and Southeast Asia. The current study focused on developing an extensive analysis method for the peripheral and integral proteins of NPC cell line HNE1. The peripheral membrane proteins were extracted by biotinylated enrichment, 0.1 M Na2CO3, and H2O. Integral or total plasma membrane fractions were prepared using 30% Percoll density grade centrifugation with or without 0.1 M Na2CO3 treatment and evaluated by Western blot analysis. The proteins were subjected to two-dimensional electrophoresis combined with tandem mass spectrometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with tandem mass spectrometry, and shotgun analysis. We identified 371, 180, and 702 proteins from peripheral, integral, and total plasma membrane fractions, respectively. In all, 848 non-redundant proteins (534 groups) were identified. Binding, catalytic, and structural molecules were the major classes. In addition to the known cell surface markers of NPC cells, the analysis revealed 311 proteins involved in multiple cell-signaling pathways and 25 proteins in disease pathways that are characteristic of cancer cells. By searching the Differentially Expressed Protein Database (http://protchem.hunnu.edu.cn/depd/index.jsp), 199 proteins were found to be differentially expressed in previous cancer proteome research. A 671 protein-protein interaction network was obtained, including 178 identified proteins in this work. The plasma membrane localization of five proteins was confirmed by immunological techniques, validating this proteomic strategy. Our study could offer some help for understanding the molecular mechanism of NPC.