Shank3 is a multi‐domain, synaptic scaffolding protein that organizes proteins in the postsynaptic density of excitatory synapses. Clinical studies suggest that ∼0.5% of autism spectrum disorder (ASD) cases may involve SHANK3 mutation/deletion. Patients with SHANK3 mutations exhibit deficits in cognition along with delayed/impaired speech/language and repetitive and obsessive/compulsive‐like (OCD‐like) behaviors. To examine how mutation/deletion of SHANK3 might alter brain function leading to ASD, we have independently created mice with deletion of Shank3 exons 4‐9, a region implicated in ASD patients. We find that homozygous deletion of exons 4‐9 (Shank3^e4‐9 KO) results in loss of the two highest molecular weight isoforms of Shank3 and a significant reduction in other isoforms. Behaviorally, both Shank3^e4‐9 heterozygous (HET) and Shank3^e4‐9 KO mice display increased repetitive grooming, deficits in novel and spatial object recognition learning and memory, and abnormal ultrasonic vocalizations. Shank3^e4‐9 KO mice also display abnormal social interaction when paired with one another. Analysis of synaptosome fractions from striata of Shank3^e4‐9 KO mice reveals decreased Homer1b/c, GluA2, and GluA3 expression. Both Shank3^e4‐9 HET and KO demonstrated a significant reduction in NMDA/AMPA ratio at excitatory synapses onto striatal medium spiny neurons. Furthermore, Shank3^e4‐9 KO mice displayed reduced hippocampal LTP despite normal baseline synaptic transmission. Collectively these behavioral, biochemical and physiological changes suggest Shank3 isoforms have region‐specific roles in regulation of AMPAR subunit localization and NMDAR function in the Shank3^e4‐9 mutant mouse model of autism. Autism Res 2016, 9: 350–375. © 2015 International Society for Autism Research, Wiley Periodicals, Inc.