Astroviruses, members of the family , represent an important cause of human gastroenteritis in the world. The cellular factors required for astrovirus replication have been poorly studied. In this work, we evaluated the relevance of the ubiquitin-proteasome system (UPS) in the replication of Yuc8, a human astrovirus serotype 8 strain. We found that proteasome inhibitors decrease the production of infectious viral progeny at a step in the replication cycle subsequent to virus entry. The inhibition of the proteasome activity decreases viral RNA levels and viral protein synthesis; similarly, the inhibition of ubiquitination by chemical inhibitors or RNAi reduces the production of viral progeny as well as viral protein synthesis. The effect on viral progeny production induced by proteasome inhibitors is not explained by a reduction in the pool of monoubiquitin or the induction of early apoptosis or autophagy. Our observations are consistent with the need of the proteolytic activity of the UPS for the efficient replication of the virus and suggest that UPS is necessary for the production of genomic and subgenomic RNA but not for antigenomic RNA. Astroviruses are a major cause of gastroenteritis in young humans and animals and recently it has been associated to fatal encephalitis in humans. The role of ubiquitin proteasome system in the replication of these viruses has not been studied before. In this work, we present evidence that supports that the proteolytic activity of proteasome is necessary for efficient viral progeny production, and that this proteolytic system is required for the accumulation of both, the genomic and subgenomic viral RNAs.