Background: The 3C protease is one of the most important proteases in enteroviruses which is responsible for several cleavages on viral polyproteins and maturation processes. Some reports indicated that this protease disrupts the interferon (IFN) signaling pathway; however, another report shows that 3C protease does not have such inhibitory effects on IFN signaling. Objectives: In this study, a construction was designed consisting of 3C protease to test its effects on Interferon type I signaling pathway. Methods: The designed construction containing 3C protease was transfected to rhabdomyosarcoma (RD) cells for evaluation of its effects on interferon signaling pathway. Rhabdomyosarcoma cells were treated with Interferon for 4 hours and then the expression of two ISGs including MxA and OAS were analyzed by real-time PCR. All the proteins in interferon type I signaling pathway were also evaluated byWestern blot analysis. Results: The results showed that 3C protease does not affect ISG expression after IFN induction. Western blot analysis also indicated that there is no difference in IFN pathway protein level between 3C protease treated and control groups. Conclusions: This report suggests that 3C protease alone does not have a significant effect on IFN pathway, in contrast to any of the previous reports.