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Background: Angiogenin (ANG) can translocate to the target cell nucleus and accumulate in the nucleolus to enhance rRNA transcription, thus promoting cell proliferation. However, the regulation of ANG-enhanced rRNA transcription remains unknown. Previously we identified phospholipid scramblase 1 (PLSCR1) as a potential ANG-interacting protein in yeast two-hybrid screening. Methods: The interaction was re-confirmed in yeast cells and further verified by in vitro pull down, in vivo co-immunoprecipitation (Co-IP), fluorescent resonance energy transfer (FRET) and immunofluorescence analyses. The rRNA transcription level was determined by real-time quantitative PCR and Northern blot. Results: PLSCR1 was identified as a novel ANG-interacting protein. Notably, PLSCR1 interacted with ANG in the cell nucleus and regulated rRNA transcription. Furthermore, depletion of cellular ANG expression abolished PLSCR1-enhanced rRNA transcription, which could be rescued by exogenous ANG. Conclusion: Our data suggest that PLSCR1 positively regulates rRNA transcription through interacting with ANG, thus deepening our understanding on rRNA transcription regulation.