Shank3 is an excitatory postsynaptic scaffolding protein implicated in multiple brain disorders, including autism spectrum disorders and Phelan-McDermid syndrome. Although previous studies of various Shank3-mutant mice have revealed diverse roles of Shank3 in the regulation of synaptic, neuronal and brain functions, whether Shank3 expression in excitatory and inhibitory neurons differentially contributes to mouse phenotypes remains largely unclear. In the present study, we generated mice carrying Shank3 deletions (exons 14–16) restricted to glutamatergic (excitatory) or GABAergic (inhibitory) neurons and characterized their electrophysiological and behavioral phenotypes. These mice show differential changes in excitatory synaptic transmission in the medial prefrontal cortex and dorsolateral striatum as well as changes in behaviors, including social interaction and communication, repetitive behaviors, locomotion and anxiety-like behaviors, with Shank3 deletion in GABAergic neurons generally inducing stronger changes. Our results suggest that glutamatergic and GABAergic Shank3 (exons 14–16) deletions differentially influence synaptic transmission and behaviors in mice.