Updated project metadata. Vaccinia related kinase (VRK) family is a ser/threonine kinase. VRK1 and VRK3 are localized in the same organelle, mainly in the nucleus. Comparative interactomes of VRK1 and VRK3 were not performed yet. We identified over 200 unreported VRK1/3-interacting proteins by proteomics-based analyses using the complementary mass spectrometry approach. Among those proteins, nuclear and cytoplasmic proteins were mostly enriched reflecting nuclear and cytoplasmic localization of VRK1/3. The interaction networks of interactome indicated biological processes associated with cell cycle, DNA repair, chromatin assembly and RNA processing. Interactions and co-localization of NCL, PARP1, HSP70, XRCC5, p53, NPM1, BAF1, cyclinB1 and ALYREF with VRK1/3 were confirmed by co-immunoprecipitation and immunofluorescent staining. Overall, this study represents a valuable resource for investigating VRK1 and VRK3 functions in health and disease, encompassing emerging themes of VRK1/3 regulation in cell cycle, mRNA processing, chromatin assembly and DNA repair.