The regulation of higher-order chromosome structure is central to cell division and sexual reproduction. Heterochromatin assembly at the centromeres facilitates both kinetochore formation and sister chromatid cohesion, and the formation of specialized chromatin structures at telomeres serves to maintain the length of telomeric repeats, to suppress recombination, and to aid in formation of a bouquet-like structure that facilitates homologous chromosome pairing during meiosis. In fission yeast, genes encoding the Argonaute, Dicer, and RNA-dependent RNA polymerase factors involved in RNA interference (RNAi) are required for heterochromatin formation at the centromeres and mating type region. In this study, we examine the effects of deletions of the fission yeast RNAi machinery on chromosome dynamics during mitosis and meiosis. We find that the RNAi machinery is required for the accurate segregation of chromosomes. Defects in mitotic chromosome segregation are correlated with loss of cohesin at centromeres. Although the telomeres of RNAi mutants maintain silencing, length, and localization of the heterochromatin protein Swi6, we discovered defects in the proper clustering of telomeres in interphase mitotic cells. Furthermore, a small proportion of RNAi mutant cells display aberrant telomere clustering during meiotic prophase. This study demonstrates that the fission yeast RNAi machinery is required for the proper regulation of chromosome architecture during mitosis and meiosis.