Background and Purpose: We previously found that secoemestrin C, an epitetrathiodioxopiperazine isolated from Aspergillus nidulans, has a potent immunosuppressive effect on splenocyte proliferation in drug screening. Here, we determined the immunomodulatory and hepatoprotective effects of secoemestrin C in a mouse model of acute autoimmune hepatitis. Experimental Approach: In an in vitro assay, purified hepatic mononuclear cells (MNCs) from C57BL/6J mice were stimulated with concanavalin A (Con A, 2 μg·mL-1) in the presence of secoemestrin C, and cell proliferation and cytokine production were measured. In an in vivo assay, mice with or without secoemestrin C pretreatment were injected with Con A (12 mg·kg-1) to induce acute hepatitis. Blood samples and liver tissues were harvested 8 h after Con A injection. Liver injury, serum levels of proinflammatory cytokines, hepatic lymphocyte subset ratios, and the functional status of NKT and conventional T cells were analyzed. Key Results: Secoemestrin C treatment dose-dependently suppressed cell proliferation and proinflammatory cytokine secretion in Con A-stimulated hepatic MNCs in vitro. In Con A-challenged mice, pre-injection with secoemestrin C significantly decreased the generation of proinflammatory cytokines and ameliorated liver injury. Furthermore, pretreatment with secoemestrin C significantly inhibited the Con A-induced activation of NKT and conventional T cells and decreased the production of IFN-γ by these two cell populations. Conclusion and Implications: Secoemestrin C has an immunosuppressive effect on NKT and conventional T cells and has hepatoprotective activity in mouse autoimmune hepatitis. These findings provide new insights into the use of fungus-derived natural products for the treatment of autoimmune diseases.