Although much is known about the oncogenic functions of chimeric Ewing sarcoma (EWS) fusion proteins that result from chromosomal translocations, the cellular role of the normal EWS protein is not well characterized. We have previously identified a WD domain-containing protein, serine-threonine kinase receptor-associated protein (STRAP), which inhibits transforming growth factor β (TGF-β) signaling through interaction with receptors and Smad7 and promotes growth and enhances tumorigenicity. Here, we report the interaction between STRAP and EWS using matrix-assisted laser desorption/ionization, time-of-flight and tandem mass spectrometry. Although STRAP is localized in both cytoplasm and nucleus, nuclear STRAP colocalizes and associates specifically with EWS in the nucleus through its NH2 and COOH termini. We have found that normal EWS protein is up-regulated in human cancers, which correlates with the up-regulation of STRAP in 71% of colorectal cancers and 54% of lung cancers, suggesting a cooperative role of these two proteins in human cancers. TGF-β has no effect on STRAP and EWS interaction. However, EWS, like STRAP, attenuates TGF-β-dependent transcription. STRAP inhibits EWS-dependent p300-mediated transactivation of EWS target genes, such as ApoCIII and c-fos, in a TGF-β-independent manner. Interestingly, we have shown that STRAP blocks the interaction between EWS and p300, whereas the complex formation between STRAP and EWS is not affected by p300. These results suggest that STRAP inhibits the transactivation function of EWS by displacing p300 from the functional transcriptional complex. Thus, this study provides a novel TGF-β- independent function of STRAP and describes a mechanism by which STRAP regulates the function of oncogenic EWS protein. ©2006 American Association for Cancer Research.