Traditionally, the multibasic cleavage site (MBCS) of surface protein H5-hemagglutinin (HA) is converted to a monobasic one so as to weaken the virulence of recombinant H5N1 influenza viruses and to produce inactivated and live attenuated vaccines. Whether such modification benefits new candidate vaccines has not been adequately investigated. We previously used retroviral vectors to generate wtH5N1 pseudotypes containing the wild-type HA (wtH5) from A/swine/Anhui/ca/2004 (H5N1) virus. Here, we generated mtH5N1 pseudotypes, which contained a mutant-type HA (mtH5) with a modified monobasic cleavage site. Groups of mice were subcutaneously injected with the two types of influenza pseudotypes. Compared to the group immunized with wtH5N1 pseudotypes, the inoculation of mtH5N1 pseudotypes induced significantly higher levels of HA specific IgG and IFN-γ in immunized mice, and enhanced protection against the challenge of mouse-adapted avian influenza virus A/Chicken/Henan/12/2004 (H5N1). This study suggests modification of the H5-hemagglutinin MBCS in retroviral pseudotypes enhances protection efficacy in mice and this information may be helpful for development of vaccines from mammalian cells to fight against H5N1 influenza viruses.