The Siah1 and Siah2 E3 ubiquitin ligases play an important role in diverse signaling pathways and have been shown to be deregulated in cancer. The human Siah1 and Siah2 isoforms share high sequence similarity but possess contrary roles in cancer， with Siah1 more often acting as a tumor suppressor while Siah2 functions as a proto-oncogene. The different function of Siah1 and Siah2 in cancer is likely due to the ubiquitination of distinct substrates. Hence， we decided to investigate the molecular basis of the substrate specificity， utilizing the well-characterized Siah2 substrate PHD3. Using chimeric and mutational approaches， we identified critical residues in Siah2 that promote substrate specificity. Thus， we have found that four residues in the N-terminal region of the Siah2 substrate binding domain (SBD) (Ser132， His150， Pro155， Tyr163) are critical for substrate specificity. In the C-terminal region of the SBD， a single residue， Leu250， was identified to promote the specific binding of Siah2 SBD to PHD3. Our study may help to overcome the challenges in the identification of Siah2 specific inhibitors.