Influenza A viruses (IAVs) are common pathogens of birds that occasionally establish endemic infections in mammals. The processes and mechanisms that result in IAV mammalian adaptation are poorly understood. The viral non-structural 1 (NS1) protein counteracts the interferon (IFN) response, a central component of the host-species barrier.We characterised the NS1 proteins of equine influenza virus (EIV), a mammalian IAV lineage of avian origin. We showed that evolutionary distinct NS1s counteract the IFN response using different and mutually exclusive mechanisms: while the NS1s of early EIVs block general gene expression by binding to the cellular polyadenylation specific factor 30 (CPSF30), NS1s from more evolved EIVs specifically block the induction of IFN-stimulated genes by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially and became rapidly selected during EIV evolution, highlighting the importance of evolutionary processes on immune evasion mechanisms during IAV adaptation. [150 words]IMPORTANCE Influenza A viruses (IAVs) infect certain avian reservoir species, and occasionally transfer to and cause epidemics of infections in some mammalian hosts. However, the processes by which IAVs gain the ability to efficiently infect and transmit in mammals remains unclear. H3N8 equine influenza virus (EIV) is an avian-origin virus that has successfully established a new lineage in horses in the early 1960, and is currently circulating worldwide in the equine population. Here we analysed the molecular evolution of the virulence factor non-structural protein 1 (NS1) and show that NS1s from different time periods after EIV emergence counteract the host innate immune response using contrasting strategies, which are associated with two mutations that appeared sequentially during EIV evolution. The results shown here indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation.