AbstractBlack widow spider is one of the most poisonous spiders in the world. Up to now, there have been few systematic analyses of the spider venom components, and the mechanism of action of the venom has not been completely understood. In this work, we employed combinative proteomic strategy to analyze the venom collected from living adult spider Latrodectus tredecimguttatus by electrical stimulation. The experiments demonstrated that the venom is primarily composed of high molecular weight proteins and has high abundance proteins around 100 kDa. The content of peptides and proteins with low molecular weight is low. A total of 75 nonredundant venom proteins with distinct function were unambiguously identified. Besides the known black widow spider venom proteins including latrotoxins, a variety of hydrolases and other proteins with special activity were found in the venom, such as proteinase, phospholipase, phosphatase, nuclease, fucolectin, venom allergen antigen 5‐like protein and trypsin inhibitor, and so on. Their possible biological actions and relationship with latrodectism were discussed. The results help to understand the complexity and action mechanism of L. tredecimguttatus venom. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:328–336, 2008; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20244