Multiple sclerosis (MS) is a chronic, autoimmune and neurodegenerative disease in which demyelination sporadically and repeatedly occurs in the central nervous system (CNS). The activity of nuclear factor kappa B (NF-κB), a family of transcription factors, was increased in the cerebrospinal fluid (CSF) and/or the serum and brain and/or spinal cord of MS patients than in a healthy donors. In our study, we investigated whether piperlongumine (PL), which is known to have inhibitory effect on activity of NF-κB, can alleviate an experimental autoimmune encephalomyelitis (EAE). The mice were immunized with myelin oligodendrocyte glycoprotein 35–55 (MOG35–55), and then we injected PL (1.5 mg/kg/day or 3.0 mg/kg/day) into the mice intraperitoneally on every second day from days 2 to 28. For in vitro study, we treated PL (0.5, 1 and 2.5 μM) to RAW 264.7 and Jurkat cells with each stimulator. We observed that the paralytic severity and neuropathology of EAE in PL-treated group were decreased compared with the EAE group. PL showed a suppressed effect on demyelination, immune cells infiltration, astrocytes/microglials activation, level of inflammatory cytokines and proteins as well as NF-κB activity. Production of inflammatory cytokines and proteins as well as translocation of NF-κB into nucleus by treatment stimulators in RAW 264.7 and Jurkat cells were reduced by PL. Moreover, treatment of NF-κB inhibitor further inhibited production of inflammatory cytokines and proteins. These results suggest that PL can mitigate MOG-induced EAE symptoms and activation of macrophages and T cells by inhibiting NF-κB signaling. Therefore, PL could be useful for the treatment for MS.