Signal transducer and activator of transcription 3 (STAT3), which is activated by cytokines and growth factors, mediates biological actions in many physiological processes. In a previous study, we found that Y14, a core component of the exon-junction complex (EJC) bound to STAT3 and upregulated the transcriptional activity of STAT3 by influencing its DNA-binding activity. In the present study, we demonstrate that STAT3 endogenously interacts with Y14. In addition, we found that MAGOH, a Y14 partner in the EJC, inhibits the STAT3-Y14 complex formation. Furthermore, small-interfering RNA-mediated reduction of MAGOH expression enhanced interleukin-6-induced gene expression. These results indicate that MAGOH regulates the transcriptional activation of STAT3 by interfering complex formation between STAT3 and Y14.