Regulation of the hematopoietic transcription factor PU.1, a member of the ETS family, plays a critical role in the development of blood cells and in leukemia. The dosage of PU.1 has been shown to cause a shift in myelomonocytic progenitor fate. Pin1 is a unique substrate‐specific enzyme that can isomerize phospho‐Ser/Thr‐Pro peptide bonds, accelerating the conformational change in its substrates between a cis and a trans form. Such activity has been demonstrated to be a tightly controlled mechanism regulating a wide variety of protein functions under both normal physiological and pathological conditions. We have previously reported that a conformational change in Runx2 induced by Pin1 is essential for its function in osteogenesis in vitro and in vivo. In this study, we show that the Pin1‐mediated conformational change in Runx1 enhances its acetylation and stabilization and, consequently, enhances its transacting activity. The increased acetylation of Runx1 represses PU.1 transcription in pre‐monocytes. Conversely, the lack of (or the inhibition of) Pin1 increases PU.1 transcription in vitro and in vivo in pre‐monocytes and in the spleen tissue. Pin1 KO mice have an increased CD11b+/F4/80+ cell population and F4/80 protein expression in spleen. From our data, we can conclude that the conformational change in Runx1 induced by Pin1 represses PU.1 transcription in pre‐monocytes and influences the commitment to the monocyte lineage. The dosage of PU.1 is a crucial factor in AML (Acute myeloid leukemia), and Pin1 may thus be a useful target for controlling PU.1‐dependent hematopoiesis, as well as leukemogenesis. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.