Emerging influenza viruses pose an extreme global risk to human health, resulting in an urgent need for effective vaccination against influenza infection. Adjuvants are vital components that can improve vaccine efficacy, yet only a few adjuvants have been licensed in human vaccines. Here, we investigate the adjuvant effects of Escherichia coli-produced monophosphoryl lipid A (MPL), named EcML, in enhancing the immunogenicity and efficacy of an influenza vaccine. Similar to MPL, EcML activated dendritic cells and enhanced the antigen processing of cells in vitro. Using ovalbumin (OVA) as a model antigen, EcML increased OVA-specific antibody production, cytotoxic T lymphocyte activity. The safety of EcML was demonstrated as being similar to that of MPL by showing not significant in vitro cell cytotoxicity but transient systemic inflammatory responses within 24 h in OVA immunized mice. Importantly, mice vaccinated with pandemic H1N1 (pH1N1) vaccine antigen, combined with EcML, were fully protected from pH1N1 virus infection by enhanced influenza-specific antibody titers, hemagglutination inhibition titers, and IFN-γ- secreting cells. Taken together, our results strongly suggest that EcML might be a promising vaccine adjuvant for preventing influenza virus infection.