Influenza virus nucleoprotein (NP) is a promising target for universal influenza vaccines due to its conservation and high immunogenicity. Here, we uncovered a previously unknown factor that E. coli-produced NP carries bacterial RNA, which is crucial for its high immunogenicity but may pose safety and consistency concerns due to batch variability. To address these concerns, we developed a NP mutant (NPmut) that lacks RNA binding activity but can be loaded with CpG1826, a synthetic oligodeoxynucleotide adjuvant that has been used in the FDA-approved Hepatitis B vaccine. The CpG1826-loaded NPmut induced immune responses comparable to RNA-bound NP while eliminating safety risks. Additionally, the mixture of CpG1826-loaded NPmut and 3M2e protein (three tandem copies of the ectodomain of influenza M2 protein) provided enhanced protection against influenza viruses challenge. Our findings highlight the adjuvant activity of bacterial RNA in E. coli-produced NP and propose a safer strategy for developing universal influenza vaccines.