The c-Myb transcription factor regulates cellular differentiation and proliferation and is regulated by complex mechanisms that control its repressed oncogenic activity. The transcriptional activity of c-Myb is regulated by the serine/threonine protein kinase Pim-1. Here, we show that Pim-1 is able to interact with c-Myb and the closely related transcription factor A-Myb, via direct interactions with the highly conserved Myb DNA binding domain. Pim-1 associated with Myb both in cells and in vitro, and phosphorylated the Myb DNA binding domain, suggesting that it regulates Myb protein activity by direct phosphorylation.