DNA replication is spatially and temporally regulated during S-phase. DNA 
replication timing is established in early-G1-phase at a point referred to as 
timing decision point. However, how the genome-wide replication timing domains 
are established is unknown. Here, we show that Rif1 (Rap1-interacting-factor-1), 
originally identified as a telomere-binding factor in yeast, is a critical 
determinant of the replication timing programme in human cells. Depletion of 
Rif1 results in specific loss of mid-S replication foci profiles, stimulation of 
initiation events in early-S-phase and changes in long-range replication timing 
domain structures. Analyses of replication timing show replication of sequences 
normally replicating early is delayed, whereas that normally replicating late is 
advanced, suggesting that replication timing regulation is abrogated in the 
absence of Rif1. Rif1 tightly binds to nuclear-insoluble structures at 
late-M-to-early-G1 and regulates chromatin-loop sizes. Furthermore, Rif1 
colocalizes specifically with the mid-S replication foci. Thus, Rif1 establishes 
the mid-S replication domains that are restrained from being activated at 
early-S-phase. Our results indicate that Rif1 plays crucial roles in determining 
the replication timing domain structures in human cells through regulating 
higher-order chromatin architecture.