Background: Influenza A virus is an orthomyxovirus capable of infecting humans. The virus genome consists of eight negative single-strand RNA segments that encode structural and nonstructural viral proteins. M2, a disulfide-linked homotetramer and a membrane-bound protein, is conserved among all influenza A viruses. Therefore, it is an appropriate target for the development of influenza vaccine with broad-spectrum protection. In this study, M2 protein of influenza virus A was expressed in a prokaryotic system. Methods: M2 gene of influenza virus A (New Caledonia/20/99, H1N1) was amplified by polymerase chain reaction (PCR) using specific primers. It was then digested with appropriate enzymes and cloned into the prokaryotic expression vector pQE30. The Escherichia coli (M15) competent cells were transformed with recombinant plasmid (pQE30-M2) and grown in LB broth media overnight after being induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The media were supplemented with 50 mg/ml ampicillin and 50 mg/ml kanamycin. Expression of the M2 protein was approved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. Findings: The results of colony PCR, restriction enzyme digestion, and sequencing revealed that M2 gene was cloned in pQE30 properly in frame to histidine tag. Protein expression was confirmed in western blotting using specific monoclonal anti-M2 antibody. Conclusion: Experiments in animal models have shown M2-based vaccines to induce broad spectrum immunity against various influenza A viruses. Thus, the M2 protein prepared in this study will be a suitable vaccine candidate to be evaluated in further studies on animal models. Keywords: M2 protein, Vaccine, Protein expression, Influenza virus, Escherichia coli