Acute liver injury (ALI) is characteristic of abrupt hepatic dysfunction and inflammatory response. Activaion of Kupffer cells (KCs) plays a central role in the pathogenesis of ALI. Since the High Mobility Group A protein2 (HMGA2) occurs as a driver at critical stage of hepatocellular carcinoma, herein, we investigated the role of HMGA2 in macrophage activation during ALI. Our study found that the expression of HMGA2 decreased dramatically both in KCs isolated from the liver in mice with ALI and in LPS-induced RAW264.7 cell lines. Moreover, loss- and gain-of-function studies suggested that HMGA2 could enhance the expression of pro-inflammatory cytokines including TNF-α, IL-6 and IL-1β. These results indicated that HMGA2 may play an essential role in macrophage activation during ALI. Additionally, our results showed the expression of HMGA2 was up-regulated when LPS-induced RAW264.7 cells were treated with 5-aza-2-deoxycytidine. Furthermore, silencing of DNMT1, DNMT3a, DNMT3b could respectively prevent the down-expression of HMGA2 in LPS-induced RAW264.7 cells. In conclusion, HMGA2 promotes the release of pro-inflammatory cytokines through NF-κB pathway, and the dysregulation of HMGA2 may involve with hypermethylation.