Der p 23 induces a high-frequency sensitization in allergic individuals. However, its allergenic activity and clinical impact are scarce. We aimed to evaluate the ability of rDer p 23 to induce allergic inflammation in a mouse model and to test IgE reactivity in humans. Female Balb/c mice were sensitized and challenged with rDer p 23 and Dermatophagoides pteronyssinus extract. Specific antibodies were determined by ELISA, inflammatory cell infiltration and goblet cells hyperplasia were evaluated by lung histology, and bronchial hyperreactivity (BHR) was assessed by the FinePoint RC System^TM and whole-body plethysmography (WBP). IgE reactivity was evaluated by ELISA, the basophils activation test (BAT) and the skin pick test (SPT) in humans. rDer p 23, produced in Escherichia coli, adopts a random coil structure, predominantly exists in a monomeric state, and exhibits high stability. rDer p 23-treated mice showed a significant increase in lung resistance and bronchial hyperreactivity, as well as in eosinophils, neutrophils, and T cell count in bronchoalveolar lavage fluid (BALF). Cytokine and antibodies profiles were biased to a Type-2 response. No significant difference was observed in group 2 Innate Lymphoid Cells (ILC-2s) in lung and regulatory T cells (Treg) in the spleen. In asthmatic individuals sensitized to D. pteronyssinus, serum IgE reactivity to rDer p 23 was 67.5%. BAT and SPT results were significantly higher in allergic patients. Our findings support the pro-allergenic role of rDer p 23 in the development of the pathological features of asthma.