Background: Clostridium perfringens and Shiga toxin (Stx)-producing Escherichia coli (STEC) are common causes of food poisoning. We previously demonstrated the efficacy of Stx2B-C-CPE, a fusion protein of the C-terminal region of C. perfringens enterotoxin (C-CPE) and Shiga toxin 2 B subunit (Stx2B), as a bivalent vaccine against C. perfringens and STEC infections. Methods: Here, we applied an E. coli expression system and Triton X-114 phase separation to prepare tag- and endotoxin-free Stx2B-C-CPE for use in vaccine formulations. Results: As we anticipated, endotoxin removal from the purified antigen reduced both Stx2B- and C-CPE-specific IgG antibody responses in subcutaneously immunized mice, suggesting that endotoxin contamination influences the immunological assessment of Stx2B-C-CPE. However, the combined use of aluminum and Alcaligenes lipid A adjuvants improved IgG antibody responses to the injected antigen, thus indicating the suitability of purified Stx2B-C-CPE for vaccine formulation. Conclusions: Our current findings provide important knowledge regarding the design of an effective commercial Stx2B-C-CPE vaccine.