The Ets transcription factor PU.1 is an essential regulator of normal hematopoiesis, especially within the myeloid lineage. As such, endogenous PU.1 predominantly localizes to the nucleus of mammalian cells to facilitate gene regulation. However, to date, little is known regarding the mechanisms of PU.1 nuclear transport. We found, using HeLa and RAW 264.7 macrophage cells, that PU.1 enters the nucleus via passive diffusion and active transport. The latter can be facilitated by: (i) the classical pathway requiring importin alpha and beta; (ii) the non-classical pathway requiring only importin beta; or (iii) direct interaction with nucleoporins. A group of six positively charged lysine or arginine residues within the Ets DNA-binding domain was determined to be crucial in active nuclear import. These residues directly interact with importin beta to facilitate a predominantly non-classical import pathway. Furthermore, luciferase reporter assays demonstrated that these same six amino acids are crucial for PU.1-mediated transcriptional activation of myeloid-specific genes. Indeed, these residues may represent a consensus sequence vital for nuclear import, DNA-binding and transcriptional activity of Ets family members. By identifying and characterizing the mechanisms of PU.1 nuclear import and the specific amino acids involved, this report may provide insights into the molecular basis of diseases.