Gephyrin is the main scaffolding protein at inhibitory synapses clustering glycine and GABA type A receptors. At specific GABAergic synapses, the nucleotide exchange factor collybistin recruits gephyrin to the postsynaptic membrane via interaction with phosphoinositides. However, the molecular mechanisms underlying the formation, maintenance and regulation of collybistin-dependent gephyrin clusters remain poorly understood. This study sheds light on the molecular mechanism of gephyrin cluster formation based on gephyrin self-oligomerization induced by collybistin, leading to the formation of a high-molecular weight (>5 MDa) gephyrin-collybistin complex, which is regulated in two ways: First, plasma-membrane phosphoinositides promote complex formation demonstrating their critical role in membrane targeting and stabilization of gephyrin-collybistin clusters at postsynaptic sites. Second, gephyrin phosphorylation at Ser325 abolishes complex formation with collybistin thus impairing collybistin-dependent gephyrin clustering at GABAergic synapses. Collectively, our data demonstrates a molecular mechanism for synaptic clustering of gephyrin which involves collybistin- and phosphoinositide-dependent formation of high-molecular gephyrin oligomers.