We studied the effects of radiofrequency electromagnetic fields (RF-EMFs) exposure on neuronal functions of mice. Particularly, we focused on RF-EMF effects on synaptic vesicles (SVs), which store neurotransmitters at axon terminals or synaptic boutons. C57 BL/6 mice were exposed to 835 MHz RF-EMF (4.0 W/kg SAR, for 5 h daily) and alterations in SVs at presynaptic terminals in the cerebral cortex were determined. Ultrastructure of randomly selected cortical neurons was observed using typical electron microscopy and bio-high voltage electron microscopy (Bio-HVEM) methods, which enable the estimation of the numbers and size of SVs. The density of the SVs (number /10 μm2 or 40 μm3) was significantly decreased in the presynaptic boutons of cortical neurons after RF-EMF exposure. Furthermore, qPCR and immunoblotting analyses revealed that the expression of synapsins I/II (Syns I/II) genes and proteins were significantly decreased in the cortical neurons of RF-EMF exposed mice. The present study suggested that alteration of SVs and Syn levels may result in alterations of neurotransmitters in the cerebral cortex following RF-EMF exposure.