Ailanthus altissima pollen (AAP) is considered as an emerging cause of respiratory allergy in United States, Italy and Iran. However, the allergenic composition of AAP is still unknown and has yet to be characterized. The present study aimed to identify AAP allergens using a proteomics-based approach. For this purpose, optimized AAP protein extracts were analyzed using 1D- and 2D- gel electrophoresis and confronted to twenty sera from individuals with respiratory allergy during the AAP season. Candidate allergens were detected using the serum from an allergic patient with clinical history of AAP pollinosis. IgE-binding spots were identified using MALDI-TOF/TOF mass spectrometry and database searching. According to our results, AAP extracts were rich in proteins (up to 16.25mg/ml) with a molecular-weight distribution ranging from 10 to 175kDa. Two-D electrophoresis of AAP extracts revealed 125 protein spots from which 13 were IgE reactive. These IgE-binding proteins were identified as enolase, calreticulin, probable pectate lyase 6, conserved hypothetical protein and ras-related protein RHN1-like. By our knowledge, this study is the first report identifying AAP allergens. These findings will open up further avenues for the diagnosis and immunotherapy of the AAP allergy as well as for the cloning and molecular characterization of relevant allergens.