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Melamine is an industrial chemical that can cause central nervous system disorders including excitotoxicity and cognitive impairment. Its illegal use in powdered baby formula was the focus of a milk scandal in China in 2008. One of our previous studies showed that melamine impaired glutamatergic transmission in rat hippocampal CA1 pyramidal cells. However, the underlying mechanism of action of melamine is unclear, and it is unknown if the CA3–CA1 pathway is directly involved. In the present study, a whole-cell patch-clamp technique was employed to investigate the effect of melamine on the hippocampal CA3–CA1 pathway in vitro. Both the evoked excitatory postsynaptic current (eEPSC) and the paired-pulse ratio (PPR) were recorded. Furthermore, we examined whether autophagy was involved in glutamatergic transmission alterations induced by melamine. Our data showed that melamine significantly increased the amplitude of eEPSCs in a dose-dependent manner. Inhibition of the
N
-methyl-
d
-aspartic acid receptor did not prevent the increase in eEPSC amplitude. In addition, the PPR was remarkably decreased by a melamine concentration of 5 × 10
−5
g/mL. It was found that autophagy could be activated by melamine and an autophagy inhibitor, 3-MA, prevented the melamine-induced increase in eEPSC amplitude. Overall, our results show that melamine presynaptically alters glutamatergic synaptic transmission of hippocampal CA3–CA1 synapses in vitro and this is likely associated with autophagy alteration.