Experimental animal and human studies have demonstrated that probiotic strains have beneficial effects on allergy. Here we report that the probiotic Escherichia coli Nissle 1917 strain (EcN) is able to activate DC, as shown by important cytokine synthesis together with up-regulation of membrane expression of CD40, CD80 and CD86. This EcN-induced DC activation was strictly dependent on the TLR4 signaling pathway and was also associated with stimulation of NF-κB and MAPK. We next investigated the prophylactic potential of i.n. co-administration of EcN with a recombinant form of Der p 1 (ProDer p 1) in a murine model of mite allergy. I.n. vaccinations with EcN plus ProDer p 1 prevented the subsequent allergic response following Der p 1 sensitization and airway challenge with aerosolized mite extracts through the induction of an allergen-specific IgG2a response, the prevention of specific IgE production and a strong reduction of IL-5 secretion by allergen-restimulated splenocytes. EcN alone or in combination with ProDer p 1 inhibited the development of airway eosinophilia and neutrophilia. This in vivo protective effect of EcN was, in part, mediated by TLR4 signaling. Our results suggest that EcN represents an efficient adjuvant to prevent allergic responses.