Quiescent cells play a predominant role in most organisms. Here we identify RNA interference (RNAi) as a major requirement for quiescence (G(0) phase of the cell cycle) in Schizosaccharomyces pombe. RNAi mutants lose viability at G(0) entry and are unable to maintain long-term quiescence. We identified suppressors of G(0) defects in cells lacking Dicer (dcr1 Delta), which mapped to genes involved in chromosome segregation, RNA polymerase-associated factors, and heterochromatin formation. We propose a model in which RNAi promotes the release of RNA polymerase in cycling and quiescent cells: (i) RNA polymerase II release mediates heterochromatin formation at centromeres, allowing proper chromosome segregation during mitotic growth and G(0) entry, and (ii) RNA polymerase I release prevents heterochromatin formation at ribosomal DNA during quiescence maintenance. Our model may account for the codependency of RNAi and histone H3 lysine 9 methylation throughout eukaryotic evolution.