In the present study, we investigated the mechanisms of brain derived neurotrophic factor (BDNF) in regulating cortical neuron premature synapse formation. KN-93, a specific inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), and G-protein-coupled receptor kinase interactor-1 (G1T1) siRNA were utilized, and the premature synapse formation of cortical neurons was detected under BDNF stimulation. Plasmids HA-GIT1, HA-GIT1 (ΔSLD), HA-GIT1 (S419A) and Flag-CaMKIIβ were constructed. The interaction between GIT1 and CaMKIIβ, and their influence on the premature synapse formation of BDNF-stimulated cortical neurons were examined. BDNF-stimulated cortical neurons were associated with increased premature synapse formation, the enhancement of phosphorylation for CaMKIIβ, and the combination of GIT1 and p-CaMKII(thr286). G1T1 siRNA and KN-93 inhibited premature synapse formation in cortical neurons. The interaction between GIT1 and CaMKIIβ required SLD domain and serine 419 in GIT1. BDNF-induced CaMKIIβ phosphorylation and premature synapse formation were suppressed in GIT1 (S419A) transfected cortical neurons. By interacting with CaMKIIβ, G1T1 (S419) were shown to participate in BDNF-induced premature synapse formation within cortical neurons.