Cdc7 is an evolutionarily conserved kinase that regulates S phase by promoting replication origin activation. Down-regulation of Cdc7 by small interfering RNA in a variety of tumor cell lines causes an abortive S phase, leading to cell death by either p53-independent apoptosis or aberrant mitosis. Unlike replication fork blockade, Cdc7-depleted tumor cells do not elicit a robust checkpoint response; thus, inhibitory signals preventing additional cell cycle progression are not generated. In normal fibroblasts, however, a p53-dependent pathway actively prevents progression through a lethal S phase in the absence of sufficient Cdc7 kinase. We show that in this experimental system, p53 is required for the lasting maintenance of this checkpoint and for cell viability. With this work we reveal and begin to characterize a novel mechanism that regulates DNA synthesis in human cells, and we suggest that inhibition of Cdc7 kinase represents a promising approach for the development of a new generation of anticancer agents.