Foot-and-mouth disease (FMD) is an economically important animal disease because of the speed of its transmission. Routine vaccination may not be effective for early protection in an outbreak situation. Small interfering RNA (siRNA) can be used in a rapid and effective antiviral approach. However, siRNA has limitations when used in disease prevention, such as a short duration of action. In this study, we have demonstrated that treatment with siRNA after FMD virus (FMDV) infection has an antiviral effect and could be effective in control of FMDV. We applied adenoviruses expressing siRNA both before and after FMDV infection in vitro and in vivo. Treatment after FMDV infection gave effective viral inhibition, but a combination of treatment before and after FMDV infection gave the best results in IBRS-2 cells. We obtained high survival rates in suckling mice by the use of therapeutic injections following challenge. The results of this study suggest that treatment with siRNA could enhance antiviral effects and may be helpful in the control of FMDV in an outbreak.