The gene promoter of Drosophila proliferating cell nuclear antigen (dPCNA) contains several transcriptional regulatory elements, such as upstream regulatory element (URE), DNA replication-related element (DRE, 5′-TATCGATA), and E2F recognition sites. In the present study, a yeast one-hybrid screen using three tandem repeats of DRE in dPCNA promoter as the bait allowed isolation of a cDNA encoding Cut, a Drosophila homolog of mammalian CCAAT-displacement protein (CDP)/Cux. Electrophoretic mobility shift assays showed that Cut bound to both DRE and the sequence 5′-AATCAAAC in URE, with much higher affinity to the former. Measurement of dPCNA promoter activity by transient luciferase expression assays in Drosophila S2 cells after an RNA interference for Cut or DREF showed DREF activates the dPCNA promoter while Cut functions as a repressor. Chromatin immunoprecipitation assays in the presence or absence of 20-hydroxyecdysone further showed both DREF and Cut proteins to be localized in the genomic region containing the dPCNA promoter in S2 cells, especially in the Cut case upon induction of differentiation. These results indicate that Cut functions as a transcriptional repressor of dPCNA gene by binding to the promoter region in the differentiated state, while DREF binds to DRE to promote expression of dPCNA during cell proliferation.