With the rapidly increasing use of mobile phones and their close-contact usage to the brain, there are some concerns about the possible neuronal effects induced by exposure to excessive electromagnetic radiation. Exposure to a radiofrequency electromagnetic field (RF-EMF) of 835 MHz (4.0 W/kg specific absorption rate (SAR) 5 h/day for 12 weeks) may affect hypothalamic presynaptic neurons in C57BL/6 mice. The number and size of the synaptic vesicles (SVs) in the hypothalamic presynaptic terminals were significantly decreased after RF-EMF exposure. Further, the density (SVs numbers/μm) of docking and fusing SVs in the active zones of the presynaptic terminal membrane was significantly decreased in hypothalamic neurons. The expression levels of synapsin I/II and synaptotagmin 1, two regulators of SV trafficking in neurons, were also significantly decreased in the hypothalamus. In parallel, the expression of calcium channel was significantly decreased. These changes in SVs in the active zones may directly decrease the release of neurotransmitters in hypothalamic presynaptic terminals. Therefore, we further studied the possible changes in hypothalamic function by testing the core body temperature and body weight and performed the buried pellet test. The trafficking of SVs was changed by RF-EMF; however, we could not find any significant phenotypical changes in our experimental condition.