Ribosomal RNA (rRNA) production occurs in the nucleolus and is a critical process for ribosome biogenesis which affects protein synthesis capacity and determines the cell growth. Dysregulation of nucleolar homeostasis elicits a nucleolar stress response and is related to disease etiology, indicating that the regulation of nucleolar activity is crucial and tightly coordinated. We previously reported that nucleolar protein pNO40 overexpression mediates SR family splicing factors into the nucleolus and impairs mRNA metabolism, while the function of pNO40 in nucleolar homeostasis is unclear. Here, we demonstrate that overexpression of pNO40 downregulates RNA polymerase I transcription activity, resulting in pre-rRNA synthesis reduction and induces nucleolar segregation, a hallmark of rRNA synthesis inhibition and nucleolar stress response. Moreover, co-immunoprecipitation experiments revealed that ectopically expressed pNO40 interacts with UBF, a master transcription factor involved in pre-initiation complex (PIC) (containing SL-1 complex and RNA polymerase I complex) to activate and promote RNA polymerase 1-mediated transcription, but disturbs its rDNA promoter binding ability. Collectively, our results demonstrate the role of pNO40 in rRNA biosynthesis regulation by compromising UBF function in rDNA transcription activation with subsequent rRNA synthesis inhibition. (C) 2019 Published by Elsevier Inc.