Abstract Background Dimethyl fumarate (DMF) is an oral immunomodulatory drug used in the treatment of autoimmune diseases. Here, we sought to study whether the effect of DMF can be detected using positron emission tomography (PET) targeting the 18-kDa translocator protein (TSPO) in the focal delayed-type hypersensitivity rat model of multiple sclerosis (fDTH-EAE). The rats were treated orally twice daily from lesion activation (day 0) with either vehicle (tap water with 0.08% Methocel, 200 µL; control group n = 4 (3 after week four)) or 15 mg/kg DMF (n = 4) in 0.08% aqueous Methocel (200 µL) for 8 weeks. The animals were imaged by PET using the TSPO tracer [¹⁸F]GE-180 in weeks 0, 1, 2, 4, 8, and 18 following lesion activation, and the non-displaceable binding potential (BP_ND) was calculated. Immunohistochemical staining for Iba1, CD4, and CD8 was performed in week 18, and in separate cohorts of animals, following 2 or 4 weeks of treatment. Results Using the fDTH-EAE model, DMF reduced the [¹⁸F]GE-180 BP_ND in the DMF-treated animals compared to control animals after 1 week of treatment (two-tailed unpaired t test, p = 0.031), but not in weeks 2, 4, 8, or 18 when imaged in vivo by PET. Immunostaining for Iba1 showed that DMF had no effect on the perilesional volume or the core lesion volume after 2 or 4 weeks of treatment, or at 18 weeks. However, the optical density (OD) measurements of CD4⁺ staining showed reduced OD in the lesions of the treated rats. Conclusions DMF reduced the microglial activation in the fDTH-EAE model after 1 week of treatment, as detected by PET imaging of the TSPO ligand [¹⁸F]GE-180. However, over an extended time course, reduced microglial activation was not observed using [¹⁸F]GE-180 or by immunohistochemistry for Iba1⁺ microglia/macrophages. Additionally, DMF did affect the infiltration of CD4⁺ and CD8⁺ T-lymphocytes at the fDTH-EAE lesion.