SummarySRSF1 (also known as ASF/SF2) is a non-small nuclear ribonucleoprotein (non-snRNP) that belongs to the arginine/serine (R/S) domain family. It recognizes and binds to mRNA, regulating both constitutive and alternative splicing. The complete loss of this proto-oncogene in mice is embryonically lethal. Through international data sharing, we identified 17 individuals (10 females and 7 males) with a neurodevelopmental disorder (NDD) with heterozygous germlineSRSF1variants, mostlyde novo, including three frameshift variants, three nonsense variants, seven missense variants, and two microdeletions within region 17q22 encompassingSRSF1. Only in one family, thede novoorigin could not be established. All individuals featured a recurrent phenotype including developmental delay and intellectual disability (DD/ID), hypotonia, neurobehavioral problems, with variable skeletal (66.7%) and cardiac (46%) anomalies. To investigate the functional consequences ofSRSF1variants, we performedin silicostructural modeling, developed anin vivosplicing assay inDrosophila, and carried out episignature analysis in blood-derived DNA from affected individuals. We found that all loss-of-function and 5 out of 7 missense variants were pathogenic, leading to a loss of SRSF1 splicing activity inDrosophila, correlating with a detectable and specific DNA methylation episignature. In addition, our orthogonalin silico,in vivo, and epigenetics analyses enabled the separation of clearly pathogenic missense variants from those with uncertain significance. Overall, these results indicated that haploinsufficiency ofSRSF1is responsible for a syndromic NDD with ID due to a partial loss of SRSF1-mediated splicing activity.