E-type cyclins (cyclins E1 and E2) are components of the cell cycle machinery that has been conserved from yeast to humans. The major function of E-type cyclins is to drive cell division. It is unknown whether in addition to their 'core' cell cycle functions, E-type cyclins also perform unique tissue-specific roles. Here, we applied high-throughput mass spectrometric analyses of mouse organs to define the repertoire of cyclin E protein partners in vivo. We found that cyclin E interacts with distinct sets of proteins in different compartments. These cyclin E interactors are highly enriched for phosphorylation targets of cyclin E and its catalytic partner, the cyclin-dependent kinase 2 (Cdk2). Among cyclin E interactors we identified several novel tissue-specific substrates of cyclin E-Cdk2 kinase. In proliferating compartments, cyclin E-Cdk2 phosphorylates Lin proteins within the DREAM complex. In the testes, cyclin E-Cdk2 phosphorylates Mybl1 and Dmrtc2, two meiotic transcription factors that represent key regulators of spermatogenesis. In embryonic and adult brains cyclin E interacts with proteins involved in neurogenesis, while in adult brains also with proteins regulating microtubule-based processes and microtubule cytoskeleton. We also used quantitative proteomics to demonstrate re-wiring of the cyclin E interactome upon ablation of Cdk2. This approach can be used to study how protein interactome changes during development or in any pathological state such as aging or cancer.