In eukaryotes, prereplication complexes (pre-RCs) containing ORC, Cdc6, Cdt1, and MCM2-7 are assembled on chromatin in the G1 phase. In S phase, when DNA replication initiates, pre-RCs are disassembled, and new pre-RC assembly is restricted until the following G1 period. As a result, DNA replication is limited to a single round per cell cycle. One inhibitor of pre-RC assembly, geminin, was discovered in Xenopus, and it binds and inactivates Cdt1 in S phase. However, removal of geminin from Xenopus egg extracts is insufficient to cause rereplication, suggesting that other safeguards against rereplication exist. Here, we show that Cdt1 is completely degraded by ubiquitin-mediated proteolysis during the course of the first round of DNA replication in Xenopus egg extracts. Degradation depends on Cdk2/Cyclin E, Cdc45, RPA, and polymerase α, demonstrating a requirement for replication initiation. Cdt1 is ubiquitinated on chromatin, and this process also requires replication initiation. Once replication has initiated, Cdk2/Cyclin E is dispensable for Cdt1 degradation. When fresh Cdt1 is supplied after the first round of DNA replication, significant rereplication results, and rereplication is enhanced in the absence of geminin. Our results identify a replication-dependent proteolytic pathway that targets Cdt1 and that acts redundantly with geminin to inactivate Cdt1 in S phase.