Enterotoxigenic Escherichia coli (ETEC) bacteria producing heat-stable toxin (STa) and/or heat-labile toxin (LT) are among top causes of children's diarrhea and travelers’ diarrhea. Currently no vaccines are available for ETEC associated diarrhea. A major challenge in developing ETEC vaccines is the inability to stimulate protective antibodies against the key STa toxin that is potently toxic and also poorly immunogenic. A recent study suggested toxoid fusion 3xSTaN12S-dmLT, which consists of a monomer LT toxoid (LTR192G/L211A) and three copies of STa toxoid STaN12S, may represent an optimal immunogen inducing neutralizing antibodies against STa toxin [IAI 2014, 82(5):1823–32]. In this study, we immunized mice with this fusion protein following a different parenteral route and using different adjuvants to further characterize immunogenicity of this toxoid fusion. Data from this study showed that 3xSTaN12S-dmLT toxoid fusion induced neutralizing anti-STa antibodies in the mice following subcutaneous immunization, as effectively as in the mice under intraperitoneal route. Data also indicated that double mutant LT (dmLT) can be an effective adjuvant for this toxoid fusion in mice subcutaneous immunization. Results from this study affirmed that toxoid fusion 3xSTaN12S-dmLT induces neutralizing antibodies against STa toxin, suggesting this toxoid fusion is potentially a promising immunogen for ETEC vaccine development.