Novel oncogene with kinase-domain (NOK) can activate multiple mitogenic signaling pathways including the janus kinases (JAK) and signal transducer and activator of transcription proteins (STAT). It was showed that NOK specifically and physically interacted with STAT3 in human embryo kidney 293T (HEK293T) cells. In addition, NOK could directly interact with most of the STAT3 subdomains except coiled-coil and C-terminal domains. Removing ectodomain and transmembrane domain of NOK markedly enhanced its intermolecular interaction with STAT3. Also, NOK could co-immunoprecipitate with JAK2 in vivo. Importantly, co-expression of NOK and JAK2 produced a synergistic effect on NOK-mediated STAT3 activation, while inactivating the kinase domain of JAK2 completely prevented this synergistic effect. Overall, the results indicated that NOK might complex with both STAT3 and JAK2 and activate STAT3 signaling by a JAK2-dependent mechanism.