Influenza A viruses encoding an altered viral NS1 protein have emerged as promising live attenuated vaccine platforms. A carboxy-terminal truncation in the NS1 protein compromises its interferon antagonism activity, making these viruses attenuated in the host yet still able to induce protection from challenge with wild-type viruses. However, specific viral protein expression by NS1-truncated viruses is known to be decreased in infected cells. In this report, we show that recombinant H5N1 and H1N1 influenza viruses encoding a truncated NS1 protein exhibited reduced expression levels of HA protein in infected cells when compared to wild-type viruses. This reduction of HA protein expression correlated with a reduction of HA mRNA levels in infected cells. NS1 truncation affected the expression of HA protein but not NP. This segment specificity was mapped to the terminal sequences of their specific viral RNAs. Since the HA protein is the major immunogenic component in influenza virus vaccines, we sought to restore its expression levels in NS1-truncated viruses in order to improve their vaccine efficacy. For this purpose, we generated an NS1-truncated rPR8 virus encoding a G3A/C8U "super-promoter" mutation in the HA genomic RNA segment. This strategy retained the attenuation properties of the recombinant virus but enhanced the expression level of HA protein in infected cells. Finally, mice immunized with rPR8 viruses encoding a truncated NS1 protein and G3A/C8U mutation in the HA segment demonstrated enhanced protection from wild-type virus challenge when compared to mice that were vaccinated with rPR8 virus encoding the truncated NS1 protein alone.