The study of arthropod venoms is of great interest to improve treatments against envenomations and provides a good tool to understand the nervous system, the immunological defense, blood coagulation and inflammatory responses. The honeybee is certainly one of the most studied animal venom and the elucidation of its proteome will be of major interest in viewing its impact on toxicity and allergy to stings. Up to now the knowledge about this venom is of limited impact due to the apparent paucity of the venom, which includes only a small number of peptides such as melittin and apamin, one major PLA₂ and a few more compounds without diversity in isoforms. The number of reports involving honeybees in Brazil is increasing, reaching approximately 15.000 stinging accidents in the last five years. Despite of this there is no specific treatment and there is only a partial identification of antigenic proteins from honeybee venom. The aim of this work is to identify the most abundant proteins in the honeybee venom using a proteomic approach. The venom was extracted using electric milking, solubilized and submitted to high resolution 2D-SDS-PAGE analysis. The spots from 2D-gels were excised, digested with trypsin and analyzed in a MALDI-Tof/MS; the protein identification was carried out by using the fingerprint setup of the protein engine search MASCOT. 2D gel revealed up to 90 distinct protein spots in CBB-stained gels. About twenty proteins have already been identified, from which 11 correspond to different isoforms of PLA₂. This enzyme represents about 12% of dry weight of total venom, it is the major allergen from this venom, is involved in inflammatory signaling cascades and in platelet aggregation among other functions. Some proteases were also identified, such as: serine-like proteases, a thrombin-like protein precursor and metaloproteinases, besides a protease inhibitor. These findings show that there are many important proteins present in this venom not reported previously. These findings and identification of other proteins should reveal some unknown proteins to better understand the mechanism of action of A. mellifera venom.