The 53-kDa insulin receptor substrate protein (IRSp53) is part of a regulatory network that organises the actin cytoskeleton in response to stimulation by small GTPases, promoting formation of actin-rich cell protrusions such as filopodia and lamellipodia. It had been established earlier that IRSp53 is tyrosine phosphorylated in response to stimulation of the insulin and insulin-related growth factor receptors, but the consequences of tyrosine phosphorylation for IRSp53 function are unknown. Here, we have used a variety of IRSp53 truncation and point mutants to identify insulin-responsive tyrosine phosphorylation sites on IRSp53. We have found that the C-terminal half of IRSp53 (residues 251-521) undergoes tyrosine phosphorylation in response to insulin stimulation of the insulin beta receptor or epidermal growth factor stimulation via the epidermal growth factor receptor, and that the key residue for insulin receptor-mediated phosphorylation is tyrosine 310, located in a region between the N-terminal IRSp53/MIM homology domain (IMD, residue 1-250) and the central SH3 domain (residues 374-438) that is predicted to be natively unstructured. Mutation of tyrosine 310 to phenylalanine or glutamic acid abrogates the phosphorylation in response to insulin stimulation, but not in response to stimulation of the epidermal growth factor receptor. The N-terminal IMD, which mediates dimerisation of IRSp53, is required for efficient tyrosine phosphorylation downstream of either the insulin or epidermal growth factor receptor stimulation, yet does not appear to include a tyrosine-phosphorylated site itself. Thus, we have identified tyrosine 310 as a primary site of tyrosine phosphorylation in response to insulin signalling and we have shown that although IRSp53 is tyrosine phosphorylated in response to epidermal growth factor receptor signalling, tyrosine 310 is not crucial. Furthermore, the tyrosine phosphorylation status does not appear to affect the cell morphology and production of filopod-like structures upon expression of IRSp53.