Mutation or disruption of the Shank/ProSAP family of genes is a high risk factor for autism spectrum disorders (ASDs) and intellectual disability. N-methyl-D-aspartate glutamate receptor (NMDAR) dysfunction contributes to the development of autism-like behaviors. However, the molecular mechanism of Shank-mediated NMDAR modulation is still not clear. Here, we show that the scaffold protein plenty of SH3s (POSH) directly interacts with two other scaffold proteins, PSD95 and SHANK2/3, at excitatory synapses. In POSH conditional knockout (cKO) mice, normal synaptic clustering of NMDAR/PSD-95/SHANK complex is disrupted, accompanied by abnormal dendritic spine development and glutamatergic transmission in hippocampal neurons. POSH cKO mice display profound autism-like behaviors, including impairments in social interactions, social communication, repetitive behaviors, and deficits in learning and memory. Thus, POSH clusters at the postsynaptic density (PSD) with PSD-95 and SHANK2/3 and plays important roles in the signaling mechanisms of the NMDAR/PSD-95/POSH/SHANK complex as well as in spine development and brain function.