Tyrosine phosphatase non-receptor type substrate-1 (SHPS-1), a transmembrane protein, plays a vital role in cell migration and proliferation. Our previous studies have shown that insulin-like growth factor-I (IGF-I) stimulates SHPS-1 phosphorylation, leading to recruitment of SHP-2, c-Src, Shc, and Grb2·p85 to phosphorylated SHPS-1. Assembly of this signaling complex is required for optimal stimulation of both mitogen-activated protein and phosphatidylinositol 3-kinase pathways. The main aim of the present study was to identify novel proteins that interacted with the cytoplasmic domain of SHPS-1 (SHPS-1/CD) in response to IGF-I stimulation and define the role of these interactions in mediating specific biological functions. We performed a functional proteomic screening to identify SHPS-1 binding partners using combination of mRNA display and the tandem affinity purification-tag methods. Screening identified a number of proteins not previously known to interact with phosphorylated SHPS-1/CD. These novel SHPS-1 binding partners represent several functional categories including heat shock proteins, protein kinases and phosphatases, and proteins that regulate transcription or translation. In Vivo and in vitro studies suggested that most of the proteins bound to SHPS-1 via binding to one of the four SH2 domain containing proteins, SHP-2, CTK, SUPT6H, and STAT1, that directly bound to SHPS-1. Although the binding of most of these proteins to SHPS-1 was positively regulated by IGF-I, a few were negatively regulated, suggesting differential regulation of protein complexes assembled on SHPS-1/CD in response to IGF-I. Further studies showed that truncation of SHPS-1/CD significantly impaired IGF-I-dependent AKT signal transduction and subsequent biological functions including cell survival, protein synthesis, protein aggregation, and prevention of apoptosis. The results emphasize the importance of formation of SHPS-1 signaling complex induced by IGF-I and provide novel insights into our knowledge of the role of this molecular scaffold in regulation of IGF-I-stimulated signal transduction and biological actions.