Small interfering RNAs (siRNAs) directed against poliovirus (PV) and other viruses effectively inhibit viral replication and have been developed as antiviral agents. Here, we demonstrate that a specific siRNA targeting the region between nucleotides 100–125 (siRNA-100) from the 5′-untranslated region (5′-UTR) of PV plays a critical role in inhibiting PV replication. Our data demonstrate that siRNA-100 treatment can greatly reduce PV titers, resulting in up-regulation of host microRNA-7 (miR-7), which in turn, leads to enhance inhibition of PV infection further. Moreover, our results suggest that siRNA-100 can also impair the spread of PV to uninfected cells by increasing host resistance to PV, resulting in decreasing necrosis and cytopathic effects (CPE) levels, as well as prolonging the survival of infected cells. Indeed, the active antiviral effect of siRNA-100 was potentially supplemented by the activity of miR-7, and both of them can serve as stabilizing factors for maintenance of cellular homeostasis. Results of this study identify a molecular mechanism of RNAi for antiviral defense, and extend our knowledge of the complex interplay between host and PV, which will provide a basis for the development of effective RNAi-based therapies designed to inhibit PV replication and protect host cells.