The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) oncoprotein has been shown to mediate activation of the signal transducer and activator of transcription 3 (STAT3). In the present study, we delineated the mechanism by which LMP1 stimulates STAT3 in a human nasopharyngeal carcinoma (NPC) cell line. LMP1 stimulated STAT3 Tyr 705-dependent nuclear accumulation, as well as the phosphorylation of STAT3 at both Tyr 705 and Ser 727. Treatment of cells with interleukin-6 neutralizing antibody inhibited the phosphorylation of STAT3 Tyr 705 and Ser 727. The differential phosphorylation of STAT3 was found to be a result of activation of Janus kinase 3 (JAK3) and extracellular signal-regulated kinase (ERK). The biological significance of JAK3-mediated activation of STAT3 Tyr 705 phosphorylation was further assessed by treating the cells with an inhibitor (WHI-P131) of JAK3. Inhibition of ERK activity by an inhibitor (PD98059) of MAPK/extracellular signal-regulated kinase kinase (MEK1) decreased the LMP1-induced activation of STAT3 Ser 727. Furthermore, immunohistochemical analysis showed an increased nuclear STAT3 Tyr 705 staining in LMP1-positive cells and STAT3 Tyr 705 phosphorylation related to NPC stages III and IV. Demonstration of the involvement of different kinases in LMP1-induced STAT3 activation supports the involvement of the JAK/STAT and mitogen-activated protein kinase (MAPK)/ERK signaling pathways in the regulation of STAT3 activation by LMP1.