14-3-3sigma is a potential tumor suppressor, and loss of 14-3-3sigma expression plays an important role in carcinogenesis and metastasis. To explore the possible mechanism of 14-3-3sigma in nasopharyngeal carcinoma (NPC) invasion and metastasis, targeted proteomic analysis was performed on 14-3-3sigma-associated proteins from NPC cells. As the results, 112 proteins associated with 14-3-3sigma were identified, and four 14-3-3sigma-interacted proteins: keratin 8, epidermal growth factor receptor (EGFR), small GTP-binding protein RAB7, and p53 were confirmed by coimmunoprecipitation and Western blot analysis. The 14-3-3sigma-associated proteins could be grouped into eight clusters based on their molecule functions. Protein-protein interaction (PPI) analysis indicated that 14-3-3sigma/EGFR/keratin 8 interactions may be involved in the invasion and metastasis of NPC. 14-3-3sigma/EGFR/keratin 8 could form complexes in NPC cells. 14-3-3sigma downregulation in NPC may lead to the overexpression of EGFR and keratin 8, which increases the invasion ability of NPC cells possibly by activating the downstream signal molecules and reorganizing cytoskeleton. The data suggest that the biological functions of 14-3-3sigma in NPC are diversified, and 14-3-3sigma could inhibit the in vitro invasive ability of NPC cells possibly through 14-3-3sigma/EGFR/keratin 8 interaction.