Efforts to develop an effective vaccine against ricin are focused on the engineering of attenuated and stable recombinant forms of the toxin's enzymatic A subunit (RTA). While several candidate antigens are in development, vaccine design and efficacy studies are being undertaken in the absence of a fundamental understanding of those regions of RTA that are critical in eliciting protective immunity. In this present study, we produced and characterized a collection of monoclonal antibodies (MAbs) directed against five distinct immunodominant regions on RTA, and used these MAbs to identify several key neutralizing epitopes on the toxin. Protective MAbs were directed against α-helices located in RTA folding domains 1 and 2, whereas non-neutralizing antibodies recognized random coils and loops that were primarily confined to folding domain 3. These data offer insights into the immunodominant and structural determinants on RTA that give rise to protective immunity, and for the first time provide an immunological rationale for ricin vaccine design.