In the tropics, both ascariasis and allergic diseases are highly prevalent. To better analyze the potential impact of Ascaris infection on the pathogenesis and diagnosis of allergic diseases, it is necessary to identify and characterize new allergens. In the tropics the IgE responses to Ascaris may be directed to species-specific or house dust mite (HDM) cross-reacting molecules. Al16 from A. lumbricoides is a predicted member of the SXP/RAL-2 protein family. We aimed to evaluate the physicochemical and immunological features of Al16. Recombinant Al16 (rAl16) was produced in Origami(D3) E. coli and purified by Ni-NTA and size exclusion chromatography. Physicochemical characterization included circular dichroism and Fourier transform infrared spectroscopy. Divalent cation binding activity was performed by ANS probe assay. Evaluation of allergenic activity included IgE binding (ELISA) in three populations: 256 adult asthmatics (ASA), 254 children from FRAAT cohort and 298 all-age subjects from Santa Catalina (SC), a rural setting. CD203 based-basophil activation tests (BAT) was also done in 12 Ascaris sensitized subject. Passive cutaneous anaphylaxis (PCA) mouse model was performed. Cross-reactivity with the HDMs Blomia tropicalis and Dermatophagoides pteronyssinus was evaluated by Western blot. Al16 identification in A. lumbricoides and HDM extracts was analyzed by liquid chromatography-mass spectrometry. rAl16 behaves as a monomer with an alpha helical folding. Its Ca2+ and Mg2+ binding was confirmed, as expected for an SXP/RAL-2 family member. IgE sensitization to Al16 (Cutoff 0.145) was higher in Ascaris sensitized individuals (ASA: 36% vs 50% p=0.031, FRAAT: 23% vs 44% p<0.001 and SC: 47% vs 58% p=0.05). In SC Ascaris+ subjects, positive BAT rate was 25%. rAl16 induced specific IgE production in sensitized mice and a positive PCA reaction and had not cross-reactive counterpart in HDM extracts. In contrast to the Ascaris extract, no Al16-related peptides were found in HDM. Al16, a divalent cation binding protein, is a new allergen from A. lumbricoides that has no cross-reactivity with HDM. It could be useful for diagnosing A. lumbricoides infection and component resolved diagnosis of HDM allergy in the tropics.