Dnmt1, Dnmt3a and Dnmt3b are main genes encoding DNA methyltransferases (Dnmts) which catalyze DNA methylation and regulate gene expression without changing DNA sequence. Our previous study disclosed that double knockout of Dnmt1 and Dnmt3a in forebrain excitatory neurons impaired synaptic plasticity and led to hippocampus-dependent learning and memory deficits, however the underlying synaptic mechanisms remain uncertain. In this study, we selectively knocked down the expression of Dnmt1 and Dnmt3a in primary cultured hippocampal neurons derived from embryonic Dnmt1,3a(2flox/2flox) mice by transfection with Cre-expressing virus, to study the effect of Dnmts and mediated DNA methylation on synaptogenesis and synaptic function. We found that the hippocampal neurons at 15 days in vitro (DIV15) exhibited similar size of cell body, but longer dendrites with reduced number of branches and lower density of excitatory synapses formation after virus-mediated Dnmt1 and Dnmt3a deletion. Supportively, cultured neurons with Dnmt1 and Dnmt3a deficiency displayed reduced frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs), indicating that both pre-and post-synaptic dysfunction are involved. In addition, our Ca2+-image study with Rhod-3AM revealed suppression of glutamate-evoked elevation of cytoplasmic [Ca2+] after Dnmt1 and Dnmt3a deletion. Altogether our findings provide new evidence that normal expression of Dnmt1 and Dnmt3a in hippocampal neurons are essential for excitatory synaptogenesis and synaptic function. (C) 2020 Elsevier Inc. All rights reserved.