Streptococcus pyogenes, the aetiological agent of both respiratory and skin infections, produces numerous exotoxins to establish infection. This report identifies a new exotoxin produced by this organism, termed SpyA, for S. pyogenesADP‐ribosylating toxin. SpyA, MW 24.9, has amino acid identity with the ADP‐riboslytransferases (ADPRTs) Staphylococcus aureus EDIN and Clostridium botulinum C3. Recombinant SpyA was able to hydrolyse β‐NAD⁺, and this activity was dependent on a glutamate at position 187. SpyA has a putative biglutamate active site, and similar to most biglutamate ADPRTs, was able to ADP‐ribosylate poly‐l‐arginine. SpyA modified numerous proteins in both CHO and HeLa cell lysates. Two‐dimesional gel analysis and MALDI‐TOF MS analysis of modified proteins indicated that vimentin, tropomyosin and actin, all cytoskeletal proteins, are targets. Expression of spyA in HeLa cells resulted in loss of actin microfilaments. We hypothesize that SpyA is produced by S. pyogenes to disrupt cytoskeletal structures and promote colonization of the host.