High mobility group box protein 1 (HMGB1) acts as a potent proinflammatory cytokine that involves in the pathogenesis of diverse inflammatory and infectious disorders. In previous study， we identified a homolog of HMGB1 in the Lampetra japonica(L-HMGB1)， and further revealed that L-HMGB1 was able to induce the production of tumor necrosis factor-α (TNF-α) in activated human acute monocytic leukemia cells. However， the role of L-HMGB1 played in lamprey was unknown. Here， we found that L-HMGB1 was located in the cytoplasm of lamprey leukocytes and supraneural body (SB) cells. Importantly， we demonstrated that L-HMGB1 participated in activation of various key molecules in inflammation signaling pathway. LPS also promoted the release of L-HMGB1 from SB cells similar to Hu-HMGB1， and then extracellular L-HMGB1 in turn induced the release of cytokines. This study revealed that the synergistic action of LPS and L-HMGB1 played a crucial role in inflammation in lamprey. Our results suggested that lampreys used L-HMGB1 to activate their innate immunity for the purpose of pathogen defense.