Influenza viral infections represent a serious public health problem, causing a contagious respiratory disease, which is most effectively prevented through vaccination. Segments seven (M) and eight (NS) of influenza virus encode mRNA transcripts that are alternatively spliced to express two different viral proteins. This study describes the generation, using reverse genetics, of three different recombinant influenza A/Puerto Rico/8/1934 (PR8) H1N1 viruses containing individual or combined modified M and/or NS viral segments in which the overlapping open reading frames of M1/M2 and/or NS1/NEP, respectively, have been split by using the porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site. Viruses with an M split segment were impaired in replication at non-permissive high temperatures, whereas high virus titers could be obtained at permissive low temperatures (33°C). Furthermore, viruses containing the M split segment were highly attenuated in vivo, while retaining immunogenicity and protection against a lethal challenge with wild-type PR8. These results indicate that influenza viruses can be effectively attenuated by the rearrangement of spliced segments and represents an excellent option as safe, immunogenic and protective live-attenuated vaccines. Moreover, this is the first time in which an influenza virus containing a restructured M segment has been described. Reorganization of M segment to encode M1 and M2 from two separate, non-overlapping, independent open reading frames represents a useful tool to independently study mutations in the M1 and M2 viral proteins without affecting the other viral M product