Objective To investigate the expression profiles of FOS protein in the mice brain subregions induced by subcutaneous formalin injection to one hindpaw. Methods After adult mice received an injection of 5% formalin in the hindpaw, their spontaneous pain behaviors were recorded within 60min. The expression, distribution and number of FOS-positive neurons in the ipsilateral dorsal horn and supraspinal regions were detected by immunohistochemical assay at two hours after formalin injection. Results These mice showed their biting/licking time and flinches in a typical two-phase manner within 60min. The immunohistochemical staining results showed that FOS-positive neurons were significantly increased in the ipsilateral spinal cord, paraventricular nucleus, medial dorsal thalamic nucleus, anterior cingulate cortex, insular cortex and amygdala. Meanwhile, the expression of FOS protein in the dentate gyrus of hippocampus was also slightly increased. Conclusion Not only spinal dorsal horn but also subregions sensitive to pain and stress, will be activated functionally to mediate defensive response when mice are exposed to nociceptive stimuli.