In the present study, an intracellular melanin (LIM) isolated from Lachnum YM30 was obtained by submerged fermentation, extraction and purification. The possible structural formula of LIM was concluded, based on Ultraviolet (UV) analysis, IR analysis, elemental analysis, Nuclear Magnetic Resonance (NMR) and ESI-MS analysis, which showed the typical feature of fungal eumelanin. The hepatoprotective effect of LIM was evaluated in an acute LPS/D-GaIN induced liver injury mouse model that was proved by the declining of alanine aminotransferase (ALT) and aspartate transaminase (AST), enhancing the content of anti-inflammatory factors interleukin-4 (IL-4) and interleukin-10 (IL-10). Concurrently, LIM could alleviate LPS/D-GaIN toxicity through the effects of NF-kappa B (NF-kappa B) signaling pathway and its related cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8). The mechanisms underlying the protective effect might be related to inhibition of nuclear factor-kappa B activation and the functional groups of LIM. And compared with the single positive drug treatment or the melanin, the results showed that the combined treatment group could further reduce the inflammatory reaction of the model mice.