PU.1, an Ets family transcription factor, was previously demonstrated expressed in 3T3-L1 preadipocytes and had an negative effect on adiopogenesis. However, the underlying mechanism remains elusive. Here, miR-191 was identified as an inhibitor of adipocyte differentiation through targeting the 3' untranslated regions of C/EBP beta, the initial factor in the C/EBR alpha/beta-PPAR gamma terminal pathway of adipogenic differentiation. MiR-191 suppressed the lipid accumulation by Oil Red O staining and downregulated the levels of adipogenic marker genes PPAR gamma (P < 0.01), aP2 (P < 0.01) and FAS (P < 0.05). Then, we found that PU.1 overexpression resulted in upregulation of miR-191 and adipogenic inhibition. Likewise. PU.1 overexpression rescued the miR-191 decrease and resisted the adipogenic promotion caused by miR-191 oligonucleotide inhibitor. Collectively, these results revealed that PU.1 promoted miR-191 to suppress adipogenesis 3T3-L1 preadipocyte and indicated a new mechanism of PU.1 inhibiting adipogenesis. (C) 2014 Elsevier Inc. All rights reserved.