Abstract DNA replication is tightly regulated to occur once per cell cycle, with the MCM2-7 helicase loaded onto replication origins only during G1-phase. In higher eukaryotes, geminin negatively regulates this process during S-, G2- and M-phases by binding the essential licensing factor CDT1. Although geminin’s function is crucial for genomic stability, its inhibitory mechanism remains elusive. Here, we utilise a fully reconstituted human DNA replication licensing assay to dissect geminin’s role.