SHIP2 is an inositol 5-phosphatase modulating the phosphatidylinositol level in plasma membrane of human cells and regulates various cell signaling pathways [1]. Notably, SHIP2 can associate with diverse phosphotyrosine (pY)-containing proteins including hepatocyte growth factor receptor c-MET, immune receptor FcgR2B and bacteria cytotoxin-related protein CagA via its SH2 domain, and is therefore related to cancers and Alzheimer’s disease [2-4]. Due to the important roles in diseases, it is crucial to understand how SHIP2-SH2 interacts with different pY-containing proteins. To this end, we analyzed the binding interface, key residues, and affinities of SHIP2-SH2 for a series of tyrsine-phosphorylated peptides derived from c-MET, FcgR2B and CagA through NMR titration and fluorescence polarization [5-7]. The results showed that a positively-charged pY-binding pocket of SHIP2-SH2 is essential for binding to all the three proteins. Meanwhile, c-MET, FcgR2B and CagA all bind to a hydrophobic area adjacent to the pY-pocket. However, the sizes of the hydrophobic area bound by the three are not equal and positively correlated with the binding affinities, which shows an order of FcgR2B, CagA, and c-MET from biggest to smallest in interface area and highest to lowest in affinity. Interestingly, FcgR2B, CagA, and c-MET contain Leu, Ile, and Val at the pY+3 site respectively, which should bind into the hydrophobic pocket of SHIP2-SH2. Thus, it was proposed that SHIP2-SH2 has a selective preference of Leu > Ile > Val at the pY+3 site in peptide ligand. In addition, the residues of FcgR2B next to the pY+3 site at C-terminal also contribute to the binding, whereas those of CagA and c-MET do not. These findings revealed the diversified binding patterns of SHIP2-SH2 for different pY-containing proteins and the sequence rule of the high-affinity peptide bound by SHIP2-SH2, which provide important clues for peptide inhibitor design.