Rabphilin 3A (Rph3A) is a synaptic protein crucial for regulating exo- and endocytosis processes at presynaptic terminals and dendritic spines. The protein promotes the stabilization of the NMDA-type glutamate receptors (NMDARs) at the cell surface, forming a ternary complex with the PSD-95 and GluN2A subunit of the receptor. This complex is needed for long-term potentiation induction and NMDAR-dependent hippocampal behaviors, such as spatial learning. Rph3A overexpression increases dendritic spine density and GluN2A-containing NMDARs at synaptic membranes. Overall, these results suggest that aberrant Rph3A could be also involved in pathological alterations of NMDAR.By trio-based exome sequencing, GeneMatcher and screening of 100,000 Genomes Project data, we identified six heterozygous variants in RPH3A gene. Four cases had a neurodevelopmental disorder (NDD) with untreatable epileptic seizures [p.(Gln73His)dn; p.(Thr450Ser)dn; p.(Arg209Lys); p.(Gln508His)], and two cases [p.(Asn618Ser)dn; p.(Arg235Ser)] showed high functioning autism spectrum disorder (ASD). Using primary hippocampal neuronal cultures, we demonstrated that p.(Arg209Lys), p.(Thr450Ser), p.(Gln508His) and p.(Asn618Ser) reduce PSD-95/GluN2A co-localization. Rph3A variants p.(Arg209Lys), p.(Thr450Ser) and p.(Gln508His) also increased the surface levels of GluN2A. These results indicate that RPH3A mutants induce GluN2A-containing NMDARs to localize prevalently at the extrasynaptic sites, suggesting that their stimulation can promote a detrimental effect. In addition, RPH3A mutants induce defects in NMDAR activity at synaptic sites, affecting postsynaptic calcium and glutamate influx at dendritic level. In conclusion, we provide evidence that missense gain-of-function variants in RPH3A increase GluN2A-containing NMDARs at extrasynaptic sites and alter synaptic function leading to a clinically variable neurodevelopmental presentation, ranging from untreatable epilepsy to ASD.