Previous studies have shown that the Epstein-Barr virus-encoded latent membrane protein1 (LMP1) could activate nuclear factor kappa B, activator protein-1, and Janus kinases/signal transducer and activation of transcription factors pathways. However, many signaling molecules and downstream target proteins triggered by LMP1 have not been identified. To determine the functional components in signaling pathways triggered by LMP1, we combined the novel strategy of phosphoprotein enrichment with proteomics technology to elucidate the signaling cascade activated by LMP1. We found that LMP1 could increase the quantity of total phosphoproteins by 18.03%, and 43 proteins showed significant changes in the degree of phosphorylation when LMP1 was expressed. Twenty-five signaling molecules or downstream targets of signaling pathways triggered by LMP1 were identified, several of which had previously been implicated in LMP1 signal pathways. The other proteins, including annexin A2, heat shock protein 27, stathmin, annexin I, basic transcription factor 3, and porin, were novel signaling molecules or targets with no previously known function in LMP1 signal transduction. The method used here has proven to be suitable for the identification of molecules involved in various signaling pathways.