To study the effects of pulse modulated radio frequency electromagnetic field on locomotor activity and neurotransmitter concentration in different brain regions of mice. Wild type C57BL/6J mice were randomly divided into intervention group (n＝9) and control group (n＝6). The intervention group was given pulse modulated RF electromagnetic field stimulation, while the control group was not given stimulation; 30 minutes a day for 5 days. The behavior of mice was recorded by camera, and the concentration of various neurotransmitters in mouse brain was measured by HPLC-MS. After the intervention, the locomotor activity of mice in the intervention group decreased every day compared with that before the intervention (P＜0.05); Intracortical concentrations of γ-aminobutyric acid, acetylcholine and other neurotransmitters, tryptophan and phenylalanine changed significantly (P＜0.05), the level of serotonin exhibited a tendency of decrease, but the concentration of glutamate did not change significantly. There were no significant changes in the locomotor activity, concentration of neurotransmitters, tryptophan and phenylalanine in the brain of control group animals. The 5-day continuous intervention had no negative effect on the anxiety level and autonomous behavior of mice. Pulse modulated RF electromagnetic field could exhibit rapid impact on the locomotor activity of mice, as well as the concentration of various neurotransmitters in different brain regions. The increased level of introcortical γ-aminobutyric acid may be involved in the reduced locomotor activity of mice following such RF electromagnetic field treatment.