Significance Voltage-gated calcium channels are important regulators of neuronal functions, as for example synaptic transmission. Their auxiliary α ₂ δ subunits are modulating the calcium currents. Beyond that they have emerged as modulators of synaptic functions. Here, we established a cellular triple knockout/knockdown model in cultured hippocampal neurons by knocking out or knocking down the expression of all three α ₂ δ subunits expressed in brain. Our experiments demonstrate that the presynaptic loss of α ₂ δ proteins leads to a severe defect in glutamatergic synapse formation, which could be rescued by reintroducing any of the three neuronal α ₂ δ isoforms. Thus, our study suggests that α ₂ δ proteins are critical regulators of excitatory synapse formation and thereby contributes to the understanding of basic nerve cell functions.