A combined analytical strategy comprising affinity enrichment, protein cross-linking and mass spectrometry enabled the identification of a total of 39 protein interaction partners of human protein kinase D2 (PKD2) from cytosolic and Golgi apparatus protein fractions. The identified binding partners revealed both already described and so far unknown functional relationships with PKD2 and provided insights into the role of PKD2 at the Golgi apparatus as well as regarding associated interactions with cytosolic effector proteins. In particular, the Arp2/3 protein complex and the AP-2 protein complex were identified, which are involved into the dynamic assembly of the cytoskeleton and the regulation of cellular vesicle transport. Furthermore, using protein cross-linking and mass spectrometric analysis of cross-linking products molecular distance constraints of PKD2 were identified. These could serve for the generation and validation of PKD2 structural models regarding the elucidation of structure-function relationships with the identified protein interaction partners.