Ret finger protein-like 1 (RFPL1) is a primate-specific target gene of Pax6, a key transcription factor for pancreas, eye and neocortex development. However, its cellular activity remains elusive. In this article, we report that Pax6-elicited expression of the human (h)RFPL1 gene in HeLa cells can be enhanced by in vivo p53 binding to its promoter and therefore investigated the hypothesis that hRFPL1 regulates cell-cycle progression. Upon expression in these cells, hRFPL1 decreased cell number through a kinase-dependent mechanism as PKC activates and Cdc2 inhibits hRFPL1 activity. hRFPL1 antiproliferative activity led to an increased cell population in G(2)/M phase and specific cyclin B1 and Cdc2 downregulations, which were precluded by a proteasome inhibitor. Specifically, cytoplasm-localized hRFPL1 prevented cyclin B1 and Cdc2 accumulation during interphase. Consequently, cells showed a delayed entry into mitosis and cell-cycle lengthening resulting from a threefold increase in G(2) phase duration. Given previous reports that RFPL1 is expressed during cell differentiation, its impact on cell-cycle lengthening therefore provides novel insights into primate-specific development.