Interneuronopathies are a group of neurodevelopmental disorders characterized by deficient migration and differentiation of GABAergic interneurons resulting in a broad clinical spectrum, including autism spectrum disorders, early-onset epileptic encephalopathy, intellectual disability, and schizophrenic disorders. SP9 is a transcription factor belonging to the Krüppel-like factor and specificity protein family, the members of which harbor highly conserved DNA binding domains. SP9 plays a central role in interneuron development and tangential migration, but it has not yet been implicated in a human neurodevelopmental disorder. Cases with SP9 variants were collected through international data-sharing networks. To address the specific impact of SP9 variants in silico and in vitro assays were carried out. De novo heterozygous variants in SP9 cause a novel form of interneuronopathy. SP9 missense variants affecting the Glutamate 378 amino acid result in severe epileptic encephalopathy due to hypomorphic and neomorphic DNA-binding effects, whereas SP9 loss-of-function variants result in a milder phenotype with epilepsy, developmental delay, and autism spectrum disorder. De novo heterozygous SP9 variants are responsible for a neurodevelopmental disease. Interestingly, variants located in conserved DNA-binding domains of KLF/SP family transcription factors may lead to neomorphic DNA-binding functions resulting in a combination of loss- and gain-of-function effects.