NANOG plays important roles in neoplastic processes. However, the molecular mechanism of NANOG in tumorigenesis remains to be elucidated. In this report, we demonstrated that forced expression of NANOG in 293 cells and cancer cells led to increased c‐Jun expression, whereas downregulation of endogenous NANOG significantly reduced c‐Jun expression in cancer cells. Dual luciferase reporter assays demonstrated that NANOG binds the c‐Jun proximal promoter and transactivates the c‐Jun gene. An ATTA consensus motif between the −160 and −268 region of the c‐Jun promoter was identified as the NANOG‐responsive element. Electromobility shift assay and chromatin immunoprecipitation results confirmed the direct binding of NANOG protein to the c‐Jun promoter in vitro and in vivo. NANOG directly bound c‐Jun protein as shown by GST pulldown and immunoprecipitation assays. Taking these findings together, we conclude that c‐Jun is a direct target gene of NANOG and that c‐Jun protein may be a novel co‐activator of NANOG in cancer cells. We suggest the possibility that NANOG may play a significant role in carcinogenesis via its activation of c‐Jun expression. © 2014 Wiley Periodicals, Inc.