Atypical antipsychotic agents, such as clozapine are used to treat schizophrenia and other psychiatric disorders by a mechanism that is believed to involve modulating the immune system. Multiple sclerosis (MS) is an immune-mediated neurological disease, and recently, clozapine was shown to reduce disease severity in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). However, the mode of action by which clozapine reduces disease in this model is poorly understood. To determine the tissue sites of clozapine action during disease, clozapine was administered to healthy and EAE mice during the time of disease development and the distribution of clozapine was monitored by MALDI mass spectrometry imaging. We found that clozapine localized to specific brain and that during EAE the distribution of clozapine was altered. Furthermore, we found that clozapine treatment reduced the infiltration of cells into the central nervous system. We assessed how the different immune cell populations were affected by clozapine treatment and the expression of chemokines was analysed in different cell types after clozapine treatment. Interestingly, we found that clozapine reduced the expression of chemokine transcripts in the brain and spinal cord which likely led to the reduced recruitment of cells into the CNS. In summary, clozapine appears to protect against EAE by multiple mechanisms involving different cell types. These results provide further information to how clozapine might be beneficial for MS patients.