Cytosine-phosphate-guanine (CpG) dinucleotide motifs are the most underrepresented motifs in RNA virus genomes, including those of the influenza A viruses (IAV). Previous studies have indicated that increasing the number of CpG motifs in IAV genomes negatively impacts viral replication. However, outcomes of these studies have been inconsistent, most likely due to variations in experimental designs. Here, the impact of altered numbers of CpG motifs in the IAV genome was investigated by carefully designing mutants with either increased or decreased frequencies of CpG motifs. Importantly, CpG mutations were excluded from regions essential for viral replication, such as predicted RNA secondary structures and alternative open reading frames, and only mutations naturally occurring in viral isolates were introduced. The resulting mutant viruses showed no significant differences in replication efficiency in human respiratory epithelial cells, nor in their ability to activate the innate immune response. Furthermore, after repeated passage in both human respiratory epithelial (A549) and MDCK cells, the introduced mutations remained stable, indicating no selection pressure on CpG motifs in vitro. This is the first study to assess the effects of genome-wide increases and decreases in CpG content in IAV while preserving essential genomic structures. Under these controlled conditions, altering CpG motif frequency did not alter the viral phenotype.IMPORTANCECpG motifs are underrepresented in RNA virus genomes, including those of influenza A virus (IAV). Previous work suggested that increasing CpG content attenuates IAV and could be exploited to design live-attenuated vaccines. However, previous work focused on single IAV genes, did not control for secondary effects, nor included mutants with decreased CpG frequencies. Here, we engineered mutants with decreased or increased CpG frequencies across multiple viral genes. Only naturally occurring mutations were introduced, while regions involved in RNA secondary structures or alternative open reading frames were preserved. Altered CpG frequencies had no significant effect on IAV replication and did not alter innate immune responses. Repeated viral passaging in immune-competent cells revealed the absence of natural selection on CpG motifs. These results demonstrate that altering CpG content does not affect IAV phenotype and challenge the rationale of using CpG enrichment to design live-attenuated IAV vaccines.