Domoic acid (DA) is an excitatory amino acids (EAAs) analog which induced excitotoxicity lesion to central nervous system, but whether induced adult animal spinal cord is not known, furthermore, previous studies have shown that EAAs play an important role in spinal cord lesion, however, the molecular pathways in spinal cord lesion are not fully known. Therefore, a motor neuron-like cell culture system and a DA-induced spinal cord lesioned mice model were used to study the effect of DA on spinal cord in adult mice and the possible molecular pathways of EAAs in spinal cord lesions. Exposure of motor neuron-like cells NSC34 to DA dramatically increased reactive oxygen species (ROS) production by the DCF fluorescent oxidation assay, reduced mitochondrial function by MTT assay, cell viability by trypan blue exclusion assay, and was accompanied by an increase of cell apoptosis by histone protein release assay. In DA-induced spinal cord lesioned mice model, we showed that the decrease of proteasome activity, increase of UCP4 expression by immunohistochemistry and neural cell apoptosis by TUNEL staining, and was accompanied by an decrease of motor disturbance grade during the different stages of DA treatment. Taken together, the in vitro and in vivo data presented in the current report demonstrated that DA induces spinal cord lesions in adult mice, and the multiple molecular pathways promoted by EAAs in spinal cord lesions, at least partially was associated with ROS generation increase, mitochondrial dysfunction, proteasome activity decrease and UCP4 expression increase.