Non-alcoholic fatty liver disease (NAFLD) is leading cause of liver failure and its negative global impact on health is growing fast. Even though there has been many efforts and studies on treating NAFLD, there is still no FDA approved medical treatment for the disease. Recently, extensive research has been carried out on G-protein coupled receptors (GPCRs)’s role in wide range of metabolic processes. Adenosine and its receptors, Adenosine a3 receptor (A3AR) have the role of regulating energy metabolism and immune response in many different diseases. Mechanistically, the activation of A3AR inhibits cAMP-PKA pathway. However, the role of how biased agonist A3AR, FM101 regulates non-alcoholic steatohepatitis and mitochondrial function remains undefined. We first measured the mRNA expression of A3AR in three major cell types in NAFLD studies: hepatocyte (HPs), hepatic stellate cell (HSCs), and Kupffer cells (KCs). Interestingly, A3AR was mostly expressed in HSCs and KCs. Then, we used FM101 to check function of A3AR activation in vitro. FM101 significantly improved inflammatory cytokines (TNF-α, IL-6, and IL-1β) and pro-fibro genic genes (COL1A1, TIMP1, and ACTA2) in KCs and HSCs, respectively. Furthermore, FM101 showed similar trend of anti-inflammatory effects compared with other NAFLD drug candidates such as OCA, UDCA and CF102. In vivo study also showed FM101’s therapeutic role with decreased liver weight and liver-to-body weight ratio. FM101 improved ALT and cholesterol level, and collagen deposition compare with the vehicle group. Through quantification of mitochondrial respiration, we found out FM101 actively block mitochondrial oxidative phosphorylation in KCs. Using MT-Keima and pMito-timer systems, FM101 treatment also induced mitochondrial vulnerability in KCs, as increased accumulation of damaged mitochondria and mitophagic degradation were observed in response to CCCP treatment. Thus, regulation of A3AR signaling could be important therapeutic target in treating NAFLD through mitochondrial mediated apoptosis pathway in KCs.