Objectives Vaccination is the most effective preventive strategy for influenza disease. As the virus undergoes high antigenic drift, it requires a constant reformulation to obtain high protection. Results Immunogenicity of a purified chimeric protein containing conserved regions of influenza A/H1N1 viruses including the Hemagglutinin stalk domain, Nucleoprotein, and Matrix protein produced in a prokaryotic system was assessed in vitro and in vivo, alone or in combination with adjuvants by evaluating antibody responses, cytokine production, lymphocyte proliferative assay, and mortality rate after challenge. The animals that received the chimeric protein had specific antibody responses, elicited memory CD4 cells, cytokines of Th1 and Th2 cells and showed 75% protection against influenza virus lethal challenge. The animals injected with the chimeric protein supplemented with Alum showed improved immune responses, but they had 67% protection. In other words, although Alum adjuvant enriched the chimera specific immune responses potently, it could not enhance its protectivity. Conclusion Regarding the immunogenicity and protectivity of the chimeric protein construct against influenza, findings of the study suggested that the chimeric protein could be considered as a promising influenza vaccine candidate.