Reactive oxygen and nitrogen species accumulate in cells during oxidative stress and cause oxidative damage to various cell components, including DNA, proteins, and lipids, thus leading to a number of severe diseases, such as atherosclerosis. Protein C3 is a central component of the complement cascade and a key player in the immune system. Proinflammatory activity of C3 can also contribute to the development of metabolic syndrome. Although hepatocytes are the main source of C3 circulating in the blood, the regulation of C3 gene expression in hepatocytes under oxidative stress remains unexplored. Suppression of C3 gene transcription and C3 protein secretion were observed during hydrogen peroxide-induced oxidative stress in HepG2 human hepatoma cells. The transcription factor FOXO1 promoted C3 expression, and C3 repression by oxidative stress was mediated through the regulation of FOXO1/HNF4α complex binding to the C3 promoter. A novel cluster of FOXO1 binding sites was identified in the distal region of the C3 promoter and found to be essential for the regulation of C3 expression by the FOXO1/HNF4α complex. Activation of the main MAP kinase cascades (ERK1/2, p38, and JNK), AMP kinase, and the transcription factor NF-κB were necessary for C3 suppression in oxidative stress. Thus, the molecular mechanisms and transcription factors that mediate suppression of C3 production in Hep G2 cells during oxidative stress were identified.