Abstract Recombinant bivalent chimeric protein was generated comprising of domain 4 of protective antigen (PA4) and carboxy terminal region of extractable antigen 1 (EA1C) by overlap extension PCR. The immunogenicity and protective efficacy of recombinant chimeric protein (PE) and protein mixture (PAEA) along with the individual components, PA4 and EA1C were evaluated in this study. We found that PE and PAEA exhibited higher endpoint titer and elevated IgG1 response. Compared to PA4 and EA1C, the chimeric protein PE and protein mixture PAEA exhibited 1.52 and 1.39 times more proliferative effect on lymphocytes in vitro. The spore uptake by anti-PE and anti-PAEA antibodies was significantly more than the individual components. We further evaluated the effects of antisera on the toxins in vitro and in vivo. Anti-PE and anti-PAEA antibodies displayed nearly 80% protection against crude toxin activity on RAW 264.7 cell lines. We further demonstrated that the anti-PE and anti-PAEA antibodies displayed better protection in controlling the edema induced by crude toxin. Passive immunization with anti-PE and anti-PAEA provided protection against toxin challenge in mice. The present study reveals that the chimeric protein consisting of heterologous regions of PA and EA1 can render better protection than PA4 or EA1C alone against toxins and bacilli.