Influenza A virus is a threat to humans due to seasonal epidemics and infrequent, but dangerous, pandemics that lead to widespread infection and death. Eight segments of RNA constitute the genome of this virus and they encode greater than eight proteins via alternative splicing of coding (+)RNAs generated from the genomic (-)RNA template strand. RNA is essential in its life cycle. A bioinformatics analysis of segment 5, which encodes nucleoprotein, revealed a conserved structural motif in the (+)RNA. The secondary structure proposed by energy minimization and comparative analysis agrees with structure predicted based on experimental data using a 121 nucleotide in vitro RNA construct comprising an influenza A virus consensus sequence and also an entire segment 5 (+)RNA (strain A/VietNam/1203/2004 (H5N1)). The conserved motif consists of three hairpins with one being especially thermodynamically stable. The biological importance of this conserved secondary structure is supported in experiments using antisense oligonucleotides in cell line, which found that disruption of this motif led to inhibition of viral fitness. These results suggest that this conserved motif in the segment 5 (+)RNA might be a candidate for oligonucleotide-based antiviral therapy.