Among other targets, the protein lysine methyltransferase PR-Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4-20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. Here, we show that H4K20 mutation in mammalian cells, unlike in Drosophila, partially impairs S-phase progression and protects from DNA re-replication induced by stabilization of PR-Set7. Using Epstein-Barr virus-derived episomes, we further demonstrate that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4-20h is not sufficient to define an efficient origin per se, but rather serves as an enhancer for MCM2-7 helicase loading and replication activation at defined origins. Consistent with this, we find that Suv4-20h-mediated H4K20 tri-methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1-associated origins, which ensure proper replication timing of late-replicating heterochromatin domains. Altogether, these results reveal Suv4-20h-mediated H4K20 tri-methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes.