Enterotoxigenic Escherichia coli (ETEC) infections have been identified as a major cause of acute diarrhoea in children in developing countries, associated with substantial morbidity and mortality rates. Additionally, ETEC remains the most common cause of acute diarrhea of international travellers to endemic areas. The heat-labile toxin (LT) is a major virulence factor of ETEC, with a significant correlation between the presence of antibodies against LT and protection in infected patients. In the present work, we constructed a recombinant LTB unit (rLTB) and studied the capacity of this toxoid incorporated in microneedles (rLTB-MN) to induce a specific immune response in mice. MN were prepared from aqueous blends of the polymer Gantrez AN® [poly (methyl vinyl ether-co-maleic anhydride)], which is not cytotoxic and has been shown to possess immunoadjuvant properties. The mechanical and dissolution properties of rLTB-MNs were evaluated in an in vitro Parafilm M® model and in mice and pig skin ex vivo models. The needle insertion ranged between 378 µm and 504 µm in Parafilm layers, and MNs fully dissolved within 15 min of application inside porcine skin. Moreover, female and male BALB/c mice were immunized through ear skin with one single dose of 5 ¿g·rLTB in MNs, eliciting significant fecal anti-LT IgA antibodies, higher in female than in male mice. Moreover, we observed an enhanced production of IL-17A by spleen cells in the immunized female mice, indicating a mucosal non-inflammatory and neutralizing mediated response. Further experiments will now be required to validate the protective capacity of this new rLTB-MN formulation against this deadly non-vaccine-preventable disease.