The common causes of Non-alcoholic fatty liver disease (NAFLD) are obesity, dyslipidemia, and insulin resistance. Metabolic disorders and lipotoxic hepatocyte damage are hallmarks of NAFLD. Even though amlexanox, a dual inhibitor of TRAF associated nuclear factor κB (NF-κB) activator-binding kinase 1 (TBK1) and IκB kinase epsilon (IKKε), has been reported to effectively improve obesity-related metabolic dysfunctions in mice models, its molecular mechanism has not been fully investigated. This study was designed to investigate the effects of amlexanox on in vitro nonalcoholic steatohepatitis (NASH) model induced by treatment of palmitic acid (PA, 0.4 mM), using a trans-well co-culture system of hepatocytes and Kupffer cells (KCs). Stimulation with PA significantly increased the phosphorylation levels of TBK1 and IKKε in both hepatocytes and KCs, suggesting a potential role of TBK1/IKKε in PA-induced NASH progression. Treatment of amlexanox (50 μM) showed significantly reduced phosphorylation of TBK1 and IKKε and hepatotoxicity as confirmed by decreased levels of lactate dehydrogenase released from hepatocytes. Furthermore, PA-induced inflammation and lipotoxic cell death in hepatocytes were significantly reversed by amlexanox treatment. Intriguingly, amlexanox inhibited the activation of KCs and induced polarization of KCs towards M2 phenotype. Mechanistically, amlexanox treatment decreased the phosphorylation of interferon regulator factor 3 (IRF3) and NF-κB in PA-treated hepatocytes. However, decreased phosphorylation of NF-κB, not IRF3, was found in PA-treated KCs upon amlexanox treatment. Taken together, our findings show that treatment of amlexanox attenuated the severity of PA-induced hepatotoxicity in vitro and lipoapoptosis by the inhibition of TBK1/IKKε-NF-κB and/or IRF3 pathway in hepatocytes and KCs.