Guillain-Barré syndrome (GBS) is an acute immune-mediated paralytic neuropathy with variable disease course and outcome. In this study, we aimed to investigate the therapeutic effects of celastrol on GBS and uncover its underlying mechanisms. Experimental autoimmune neuritis (EAN) is a typical animal model for GBS, and thus an EAN rat model was established with the injection of celastrol or/and LPS. We assessed the body weights and EAN clinical scores of rats. HE staining, flow cytometry, RT-qPCR, and Western blotting were respectively employed to measure pathological damage, proportions of cells (Th1, Th17, and Treg), Th1/Th17 cell differentiation-related mRNAs (IFN-γ, TBX21, IL-18, RORγT, IL-17, and IL-23) and TLR4/NF-κB/STAT3 pathway-related proteins (TLR4, NF-κB, p-NF-κB, STAT3, and p-STAT3). We found that celastrol attenuated clinical symptoms and pathological damage of GBS in EAN rats. Moreover, celastrol down-regulated Th1 and Th17 cell proportions, and the levels of IFN-γ, TBX21, IL-18, RORγT, IL-17, and IL-23 in EAN rats. Meanwhile, the levels of TLR4, p-NF-κB, and p-STAT3 were decreased by celastrol. Taken together, celastrol could restrain Th1/Th17 cell differentiation through inhibition of the TLR4/NF-κB/STAT3 pathway in EAN rats. Our findings suggest that celastrol may exert therapeutic effects on GBS by suppressing TLR4/NF-κB/STAT3 pathway-mediated Th1/Th17 cell differentiation.