The binding of insulin to its receptor triggers a signaling cascade regulated by protein complexes via tyrosine phosphorylation events on a multitude of associated proteins. To search novel phosphotyrosine proteins or associated proteins involved in insulin signaling pathway, we employed a method in which Rat1 cells stably expressing the human insulin receptor were stimulated with or without insulin and sub-fractionated prior to enrichment of phosphotyrosine proteins by immunoprecipitation and analysis by LC-MS/MS. Bioinformatic analysis and manual confirmation of peptide phosphorylation site assignments led to identification of 35 phosphotyrosine sites derived from 31 protein groups. Over 50% of these proteins were reported for the first time as tyrosine phosphorylated, including gigaxonin, XIAP and CDK10. In addition, we also found that calcium/calmodulin-dependent protein serine kinase (CASK), a key protein in protein-targeting and vesicle transport in neurons, forms a complex with two unidentified phosphotyrosine proteins pp100 and pp95 in response to insulin-stimulation, though CASK is not itself tyrosine phosphorylated. Furthermore, insulin was able to decrease CASK nuclear location, as well as down-regulate the expression of CASK targeted genes. Our results imply CASK as a novel joint knot connecting CASK-mediated pathways with the insulin signaling. Our data provide a wealth of information potentially paving the way to identify new components in the insulin signaling network.