Vaccination is the most effective intervention to prevent influenza and control the spread of the virus. Alternatives are needed to the traditional egg-based vaccine strategy for a more rapid response to new outbreaks. Two different hemagglutinin (HA) fragments (rHA11-326 and rHA153-269) derived from influenza A virus subtype H1N1 were expressed in Escherichia coli and characterized by immunoblot, gel filtration, hemagglutination, and competitive binding assays. rHA11-326 included neutralizing epitopes and the trimerization domain, whereas rHA153-269 included only the head of HA with the neutralizing epitopes. Mice were immunized with rHA11-326 or rHA153-269, and sera were tested for the presence of neutralizing antibodies. Mice were then challenged with H1N1 and infection severity was monitored. rHA11-326 trimerized, whereas rHA153-269 was unable to form oligomers. Both rHA11-326 and rHA153-269 elicited the production of neutralizing antibodies, but only oligomerized rHA11-326 protected against live virus challenges in mice. This study demonstrated that bacterially expressed HA was capable of folding properly and eliciting the production of neutralizing antibodies, and that HA oligomerization contributed to protection against viral challenge. Therefore, prokaryotic-derived vaccine platforms can provide antigenic and structural requirements for viral protection, as well as allow for the rapid and cost-effective incorporation of multiple antigens for broader protection.