In this study, we examined whether electroacupuncture (EA) represses pruritogen-induced microglial activation. Immunohistochemical studies revealed that a subcutaneous (s.c.) injection of the pruritogen 5′-guanidinonaltrindole (GNTI; 0.3 mg/kg) to the back of the neck in mice induced acute expression of the ionized calcium-binding adaptor molecule 1 (Iba1) in both gray and white matter of the spinal cord, with the highest expression in the dorsal horn area. EA application (2 Hz) to LI4 and LI11 attenuated GNTI-induced scratching behavior and repressed GNTI-induced Iba1 expression and Iba1 (+) microglia in the dorsal horn. In contrast, EA at the ST36 acupoint had no such effects. Confocal image analysis revealed co-expression of phosphorylated p38 and Iba1 in microglia with EA at the ST36 acupoint, but not at the LI4 or LI11 acupoints. In Western blot analysis, s.c. injection of GNTI to the back of the neck increased Iba1 and phospho-p38 expression in the spinal cord as compared with injection of saline, while EA at LI4 and LI11 reduced GNTI-induced expression of Iba1 and phospho-p38. These findings indicate that EA at LI4 and LI11, but not at ST36, reduces GNTI-induced microglial activation in the mouse spinal cord.