Rubiadin, a bioactive dihydroxyanthraquinone found in various plants, has been documented to have antioxidant activity. Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common cause of chronic liver disease, in which oxidative stress plays a pivotal role in pathological development. In this study, we aimed to investigate the therapeutic effect of Rubiadin on hepatic steatosis and its underlying mechanism. Pathological conditions of MASLD were reproduced on HepG2 cells using hydrogen peroxide or the mixture of fructose, palmitic acid (PA), and oleic acid (OA). Western blotting was used to determine the expression levels of related proteins. A reactive oxygen species (ROS) assay was performed in HepG2 cells. Rubiadin reversed overall cell viability in HepG2 cells treated with hydrogen peroxide. Western blotting results demonstrated that Rubiadin effectively recovered superoxide dismutase type 1 (SOD1) and catalase activities suppressed by hydrogen peroxide. Furthermore, Rubiadin successfully diminished ROS production triggered by hydrogen peroxide or the mixture of fructose, PA, and OA. These results suggest that Rubiadin ameliorates apoptosis and oxidative stress by restoring the activities of antioxidant enzymes. The present study provides a novel therapeutic strategy for treating MASLD using the natural compound, Rubiadin.