ABSTRACT Erythroid enucleation is the final stage of erythroid terminal differentiation and involves the separation of an orthochromatic erythroblast into two daughter cells – a pyrenocyte containing the extruded nucleus, and a reticulocyte, which will become a red blood cell. Our previous work has identified CDK9 as a regulator of erythroid enucleation that appears to act independently of its known role in regulating RNA polymerase II transcription, suggesting the potential for a new CDK9 role. Using a co-immunoprecipitation and mass spectrometry approach, we here identified the interactome of CDK9 in differentiating erythroblasts. We show that CDK9 interacts with a RanGTP–importin-β complex during erythroid terminal differentiation, and inhibition of importin-β in erythroblasts blocks erythroid enucleation. Using imaging analysis and functional assays of enucleating erythroblasts, we show that CDK9 and importin-β colocate at a crucial site of activity opposite to the nucleus before nuclear extrusion and we describe a novel finding that physically links CDK9 and importin-β activity prior to calmodulin and Ca2+ signalling, and subsequent F-actin activity, to achieve enucleation.