ABSTRACTHigh-impact pathogenic variants in more than 1,000 protein-coding genes cause Mendelian forms of neurodevelopmental disorders (NDD), including the newly reportedAGO2gene. This study describes the molecular and clinical characterization of 28 probands with NDD harboring heterozygousAGO1coding variants.De novostatus was always confirmed when parents were available (26/28). A total of 15 unique variants leading to amino acid changes or deletions were identified: 12 missense variants, two in-frame deletions of one codon, and one canonical splice variant leading to a deletion of two amino acid residues. Some variants were recurrently identified in several unrelated individuals: p.(Phe180del), p.(Leu190Pro), p.(Leu190Arg), p.(Gly199Ser), p.(Val254Ile) and p.(Glu376del).AGO1encodes the Argonaute 1 protein, which functions in gene-silencing pathways mediated by small non-coding RNAs. Three-dimensional protein structure predictions suggest that these variants might alter the flexibility of the AGO1 linkers domains, which likely would impair its function in mRNA processing. Affected individuals present with intellectual disability of varying severity, as well as speech and motor delay, autistic behavior and additional behavioral manifestations. Our study establishes thatde novocoding variants inAGO1are involved in a novel monogenic form of NDD, highly similar toAGO2phenotype.