ABSTRACT Adenosine 2A receptor (A 2A R) exerts protective roles in endotoxin- and/or ischemia-induced tissue damages. However, the role for A 2A R in non-alcoholic fatty liver disease (NAFLD) remains largely unknown. We sought to examine the effects of global and/or myeloid cell-specific A 2A R disruption on the aspects of obesity-associated NAFLD and to elucidate the underlying mechanisms. Global and/or myeloid cell-specific A 2A R-disrupted mice, as well as control mice were fed a high-fat diet (HFD) to induce NAFLD. Also, bone marrow-derived macrophages and primary mouse hepatocytes were examined for inflammatory and metabolic responses. Upon feeding an HFD, both global A 2A R-disrupted mice and myeloid cell-specific A 2A R-defcient mice revealed increased severity of HFD-induced hepatic steatosis and inflammation compared with their respective control mice. In in vitro experiments, A 2A R-deficient macrophages exhibited increased proinflammatory responses, and enhanced fat deposition of wild-type primary hepatocytes in macrophage-hepatocyte co-cultures. In primary hepatocytes, A 2A R deficiency increased the proinflammatory responses and enhanced the effect of palmitate on stimulating fat deposition. Moreover, A 2A R deficiency significantly increased sterol regulatory element-binding protein 1c (SREBP1c) abundance in livers of fasted mice and in hepatocytes upon nutrient deprivation. In the absence of A 2A R, SREBP1c transcription activity was significantly increased in mouse hepatocytes. Taken together, these results demonstrate that disruption of A 2A R in both macrophage and hepatocytes accounts for increased severity of NAFLD, likely through increasing inflammation and through elevating lipogenic events due to stimulation of SREBP1c expression and transcription activity. This article is protected by copyright. All rights reserved.