Neuronal diversity is crucial for a functioning mature brain. Identifying the molecular factors that underpin this diversity is essential to our understanding of the brain in health and disease. Forebrain embryonic zinc finger 2 (Fezf2) encodes a transcription factor essential to the specification of projection neuron fate in the developing cerebral cortex. As with many developmentally important transcription factors, Fezf2 is expressed into adulthood, suggesting it could have importance for the maintenance of mature neurons. Despite the continued expression, a function for Fezf2 in the mature brain has yet to be explored. In this work, we investigated a role for Fezf2 in mature neurons using a lentiviral approach to conditionally knockdown the expression of Fezf2 in the mature primary motor cortex (M1). RNA-seq analysis on this tissue revealed significant changes to the molecular profile of Fezf2-reduced M1 tissue, with 756 differentially expressed genes identified (FDR ≤ 0.05, LFC ≥ 0.2). Further term enrichment analysis indicated a common role for Fezf2-regulated genes in neuronal functions, including synaptic transmission and ion channel activity. We also saw significant overrepresentation of Fezf2-regulated genes implicated in sensory and motor-associated behaviour phenotypes. In a complementary study using Drosophila, we show that the conditional reduction of Fezf2 homologue, dfezl, leads to significant changes in the sensorimotor behaviour of adult Drosophila, confirming a functional role. Our results demonstrate a regulatory role for Fezf2 and contribute generally to the growing evidence that developmental transcription factors have a critical function in the mature brain.