Background. In mammalian cells regulatory proteins controlling the cell cycle are necessary due to the requirements of living in a heterogeneous environment of cell-interactions and growth factors. VRK1 is a novel serine-threonine kinase that phosphorylates several transcription factors and is associated with proliferation phenotypes. Methodology/Principal Findings. In this report VRK1 has been identified as regulated in the cell cycle. VRK1 gene expression is activated by the addition of serum to starved cells, indicating it is required for the exit of G0 phase and entry in G1; a response that parallels the re-expression of MYC, FOS and CCND1 (cyclin D1) genes, suggesting that VRK1 is an early-response gene. VRK1 gene expression is also shutdown by serum withdrawal. The human VRK1 gene promoter cloned in a luciferase reporter responds similarly to serum. In response to serum, the level of VRK1 protein expression has a positive correlation with cell proliferation markers such as phosphorylated-Rb or PCNA, and is inversely correlated with cell cycle inhibitors as p27. The elimination of VRK1 by siRNA results in a G1 block in cell division, and in loss of phosphorylated-Rb, cyclin D1, and other proliferation markers. Elimination of VRK1 by siRNA induces a reduction of cell proliferation. VRK1 colocalizes with p63 in proliferating areas of squamous epithelium, and identifies a subpopulation in the basal layer. Conclusions/Significance. VRK1 is an immediate early response gene required for entry in G1, and due to its implication in normal cell proliferation and division, might be a new target for development of inhibitors of cellular proliferation.This work was partially funded by grants from Ministerio de Educación y Ciencia (SAF2004-02900, SAF2007-60242 and CSD2007-0017), Junta de Castilla y León (CSI05A05), Federación de Cajas de Ahorro de Castilla y León and Fundación de Investigación Médica MM to P. A. Lazo. A. Valbuena and I. López-Sánchez have predoctoral fellowships from Ministerio de Educación y Ciencia.Peer reviewed