Abstract Eukaryotic cell cycle progression requires the coordination of multiple molecular events in space and time to bring about proper transition from one phase to the next, and to ensure faithful genomic inheritance to daughter cells. The roles of coding transcription, translation, and RNA/protein degradation in cell cycle regulation are straightforward, since they control the abundance of proteins necessary for its progression. However, the contribution of non-coding transcription to this process is poorly understood. Non-coding transcription is notoriously interesting in mitosis for two reasons: First, transcription is repressed in most of the genome, but it is active in the centromere itself during mitosis; second, centromeric transcription produces non-coding RNA molecules which are integral components of the centromere and kinetochore and have functional roles in chromosome segregation. Here, we characterized the centromeric human alpha satellite non-coding RNAs (cencRNAs). We detected that the centromere produces high-molecular-weight (&gt5 kb) alpha satellite transcripts in sense and antisense orientation in several cancer cell lines and normal human leukocytes. These RNAs have a half-life of ~ 30 min and vary in abundance, but not in size, throughout the cell cycle. By analyzing their abundance under various conditions that promote mitotic arrest, we discovered a new association between proteasome inhibition and cencRNAs overexpression in G2/M HCT116 cells. We also show that their transcriptional inhibition mitigates the mitotic arrest induced by proteasome inhibitors. Furthermore, we demonstrate that exogenous expression of cencRNAs can slightly increase the mitotic index of SW480 cells. Using mass spectrometry-coupled RNA pulldown, we discovered that these RNAs interact with proteins essential for chromosome segregation. Our data show that centromeric transcription is regulated by proteasomes. Proteasomal inhibition promotes abnormal mitotic progression owing to the overexpression of non-coding RNAs at the onset of mitosis and the presumed impairment of their partner proteins functions in mitosis. Citation Format: Rodrigo E. Cáceres, Marco A. Andonegui, Diego A. Oliva, Rodrigo González, Fernando Luna, Cristian G. Arriaga, Alejandro López, Diddier G. Prada, Clementina Castro, Paulina Molina, Carlos De la Rosa, José L. Reyes, Sabrine Hédouin, Florent Hubé, Claire Francastel, Luis A. Herrera. Molecular and functional characterization of alpha satellite non-coding RNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4422.