The dentate gyrus (DG) is modified throughout life by integration of new adult-born neurons. Similarities in neuronal maturation during DG development and adult hippocampal neurogenesis suggest that genetically encoded intrinsic regulatory mechanisms underlying these temporally distinct processes are conserved and reused. Here, we identify a novel transcriptional regulator of dentate granule neuron maturation, Krüppel-like factor 9 (Klf-9). We show that Klf-9 expression is induced by neuronal activity and as dentate granule neurons functionally integrate in the developing and adult DG. During development, dentate granule neurons lacking Klf-9 show delayed maturation as reflected by altered expression of early-phase markers, dendritic spine formation, and electrophysiological properties. Adult Klf-9-null mice exhibit normal stem cell proliferation and cell fate specification in the DG but show impaired differentiation of adult-born neurons and decreased neurogenesis-dependent synaptic plasticity. Behavioral analysis of Klf-9-null mice revealed a subtle increase in anxiety-like behavior and an impairment in contextual fear discrimination learning. Thus, Klf-9 is necessary for late-phase maturation of dentate granule neurons both in DG development and during adult hippocampal neurogenesis. Klf-9-dependent neuronal maturation may therefore represent a candidate regulatory mechanism underlying these temporally distinct processes.