Update information. HMGB1 has been extensively studied as a DAMP, with secreted cytokine function. However, its regulation on T cells, especially the function in the nucleus, has not been elucidated. Here, we extracted the total protein of CD8 T cells and enriched the proteins bound to HMGB1 through IP experiment. We found several proteins binding with HMGB1, which suggested that nuclear HMGB1 might regulate the function of CD8 T cells by interacting with these proteins. In our study, we focused on the binding of HMGB1 and Eomes, and found that HMGB1 supported the expression of IFN-g in CD8 T cells by directly regulating the activity of the transcription factor Eomes of IFN-g. In conclusion, we determined the role of nuclear HMGB1 in the differentiation of CD8 T cells and proved that it played an important role in the anti-tumor program of CD8 T cells.