Western equine encephalitis virus (WEEV) is a positive-sense, single-stranded RNA virus which is transmitted to equines and humans through mosquito bites. WEEV infects the central nervous system with severe complications and even death. There are no human vaccine and antiviral drugs. We investigated whether adenovirus-mediated expression of interferon alpha could be used for pre- and post-exposure protection against a lethal WEEV challenge in mice. A human adenoviral vector (Ad5-mIFNalpha) expressing mouse interferon alpha was constructed. We found that Ad5-mIFNalpha provided 100% protection against various WEEV strains in mice after a single intramuscular inoculation at 24 h, 48 h or 1 week before the challenge. When given as a single inoculation at 6 h after the challenge, Ad5-mIFNalpha delayed the progress of WEEV infection and provided about 60% protection. Our findings suggest that adenovirus-mediated expression of interferon alpha can be an alternative approach for the prevention and treatment of WEEV infection.