Cyclin-dependent kinases (CDKs) are key players in cell cycle regulation and transcription. The CDK-family member Cdk10 is important for neural development and can act as a tumor suppressor but the underlying molecular mechanisms are largely unknown. Here, we provide an in depth analysis of Cdk10 substrate specificity and function. Using recombinant Cdk10/CycQ protein complexes we characterize RNA pol II CTD, c-MYC, and RB1 as in vitro protein substrates. Using an analog-sensitive mutant kinase, we identify 66 proteins as Cdk10 substrates from HEK cells. Among these, proteins involved in cell cycle, translation, stress response, growth signalling, as well as rRNA, and mRNA transcriptional regulation are found. Cdk10 is able to auto-phosphorylate in trans within the T-loop, offering the possibility of a positive feedback loop for activation. We also identify Cdk10 as an in vitro substrate of Cdk1 and Cdk5 at multiple sites, allowing for a potential cross-talk between these CDKs. With this functional characterization, Cdk10 adopts a hybrid position in both cell cycle and transcriptional regulation