Peramivir is a neuraminidase (NA) inhibitor (NAI) under development that must be administered by the systemic route. The prophylactic activity of intramuscular (IM) peramivir was evaluated with mice infected with wild-type (WT) and oseltamivir-resistant (H274Y NA mutant) recombinant influenza A/WSN/33 (H1N1) viruses. Treatment regimens consisted of IM injections starting 1 h before viral challenge that were single (45 mg/kg or 90 mg/kg) or multiple (45 mg/kg daily for 5 days). All peramivir regimens prevented mortality and weight loss while significantly reducing lung viral titers (LVT) in mice infected with the WT virus. For animals infected with the H274Y mutant, the multiple-dose regimen completely prevented mortality and was associated with significant reduction in weight loss and LVT compared to untreated animals. In contrast, both single-treatment regimens reduced mortality and weight loss but did not significantly reduce LVT. Although further experiments using different influenza A/H1N1 virus strains and other animal models are needed, our results suggest that 5-day IM peramivir therapy may be considered a prophylactic alternative to control influenza infections caused by oseltamivir-resistant viruses with the H274Y mutation.