The structural maintenance of chromosomes (SMC) proteins constitute the core of critical complexes involved in structural organization of chromosomes. In yeast, the Smc5/6 complex is known to mediate repair of DNA breaks and replication of repetitive genomic regions, including ribosomal DNA loci and telomeres. In mammalian cells, which have diverse genome structure and scale from yeast, the Smc5/6 complex has also been implicated in DNA damage response, but its further function in unchallenged conditions remains elusive. In this study, we addressed the behavior and function of Smc5/6 during the cell cycle. Chromatin fractionation, immunofluorescence, and live-cell imaging analyses indicated that Smc5/6 associates with chromatin during interphase but largely dissociates from chromosomes when they condense in mitosis. Depletion of Smc5 and Smc6 resulted in aberrant mitotic chromosome phenotypes that were accompanied by the abnormal distribution of topoisomerase IIα (topo IIα) and condensins and by chromosome segregation errors. Importantly, interphase chromatin structure indicated by the premature chromosome condensation assay suggested that Smc5/6 is required for the on-time progression of DNA replication and subsequent binding of topo IIα on replicated chromatids. These results indicate an essential role of the Smc5/6 complex in processing DNA replication, which becomes indispensable for proper sister chromatid assembly in mitosis.