The common cypress (Cupressus sempervirens) (Cups) pollen represents the first cause of respiratory allergies in the Mediterranean basin. The aim of this study was to characterize a novel 14-kDa cypress pollen allergen (BP14) allowing a clear dissociation of IgE sensitization patterns among allergic patients. The biochemical and immunochemical characterization of BP14 included determination of its isoelectric point, molecular mass, extraction kinetics, IgE binding prevalence, the presence of bromelain-type cross-reactive carbohydrate determinant and its IgE reactivity under reducing conditions. The presence of potential cross-reactive homologues in closely related cypress species, i.e. Cupressus arizonica (Cupa) and Cryptomeria japonica (Cryj), as well as in several taxonomically unrelated species was also investigated. According to our results, BP14 is easily and quickly solubilized in phosphate-buffered saline and exhibits several allergenic isoforms covering a broad range of pI (6.5–10.5). This allergen displays heat-stable conformational epitopes and does not include cross-reactive carbohydrate determinants, in contrast to high molecular weight cypress allergens. BP14 is expressed at higher levels in Cups than in Cupa and Cryj. No IgE cross-reactivity was found between the 14-kDa Cups pollen protein and proteins from some other non-Cupressaceae pollen allergenic sources such as orchard, timothy, wheat, maize, birch, ash and pine. Thus, IgE reactivity to BP14 is specific to Cupressaceae and discriminates two groups of patients allergic to cypress pollen. It might correspond to a relevant marker in relation to the sensitization process and/or the symptoms observed in some cypress-pollen-allergic patients. Furthermore, the description of BP14 should improve the diagnosis of cypress pollinosis.