Using a cell-free system that reconstitutes initiation of mammalian DNA replication, we identified a cyclin A-responsive protein, p21(Cip1)-interacting zinc finger protein 1 (Ciz1). In cell-free experiments, Ciz1 protein increases the number of nuclei that initiate DNA replication, and in intact cells GFP-tagged Ciz1 stimulates DNA synthesis, in both a wild-type and a p21(Cip1) null background. Furthermore, mutation of a putative cyclin-dependent kinase phosphorylation site at threonines 191/2 alters Ciz1 activity in vitro, indicating that this site plays a role in regulating Ciz1. Consistent with a role in DNA replication, endogenous Ciz1 is present in nuclear foci that co-localize with PCNA during S phase, and targeted depletion of Ciz1 transcripts restrains cell proliferation by inhibiting entry to S phase. Ciz1-depleted cells accumulate with chromatin bound Mcm3 and PCNA but fail to synthesize DNA efficiently. These cell-based and cell-free experiments suggest that Ciz1 functions to promote DNA replication after replication complex formation. Finally, alternatively spliced forms of Ciz1 occur in embryonic cells from mouse and man, raising the possibility that Ciz1 splicing contributes to the regulation of DNA replication during development.