AbstractType I IFN play a very important role in immunity against viral infections. Murine type I IFN belongs to a multigene family including 14 IFN‐α subtypes but the biological functions of IFN‐α subtypes in retroviral infections are unknown. We have used the Friend retrovirus model to determine the anti‐viral effects of IFN‐α subtypes in vitro and in vivo. IFN‐α subtypes α1, α4, α6 or α9 suppressed Friend virus (FV) replication in vitro, but differed greatly in their anti‐viral efficacy in vivo. Treatment of FV‐infected mice with the IFN‐α subtypes α1, α4 or α9, but not α6 led to a significant reduction in viral loads. Decreased splenic viral load after IFN‐α1 treatment correlated with an expansion of activated FV‐specific CD8+ T cells and NK cells into the spleen, whereas in IFN‐α4‐ and ‐α9‐treated mice it exclusively correlated with the activation of NK cells. The results demonstrate the distinct anti‐retroviral effects of different IFN‐α subtypes, which may be relevant for new therapeutic approaches.