Nasal application of native cholera toxin (nCT) as a mucosal adjuvant has potential toxicity for the CNS through binding to GM1 gangliosides in the olfactory nerves. Although mutants of cholera toxin (mCTs) have been developed that show mucosal adjuvant activity without toxicity, it still remains unclear whether these mCTs will induce CNS damage. To help overcome these concerns, in this study we created new double mutant CTs (dmCTs) that have two amino acid substitutions in the ADP-ribosyltransferase active center (E112K) and COOH-terminal KDEL (E112K/KDEV or E112K/KDGL). Confocal microscopic analysis showed that intracellular localization of dmCTs differed from that of mCTs and nCTs in intestinal epithelial T84 cells. Furthermore, both dmCTs exhibited very low toxicity in the Y1 cell assay and mouse ileal loop tests. When mucosal adjuvanticity was examined, both dmCTs induced enhanced OVA-specific immune responses in both mucosal and systemic lymphoid tissues. Interestingly, although both dmCT E112K/KDEV and dmCT E112K/KDGL showed high Th2-type and significant Th1-type cytokine responses by OVA-specific CD4+ T cells, dmCT E112K/KDEV exhibited significantly lower Th1-type cytokine responses than did nCT and dmCT E112K/KDGL. These results show that newly developed dmCTs retain strong biological adjuvant activity without CNS toxicity.