In this study, the mechanism that the inhibition of glycogen synthase kinase-3β (GSK-3β) promotes the production of reactive oxygen species (ROS) via β-catenin/CCAAT/enhancer binding protein α (C/EBPα) signaling was investigated in the spleen of zebrafish (Danio rerio). The results demonstrated that the inhibition of GSK-3β induced the mRNA expression of β-catenin and C/EBPα by lithium (Li) treatments or GSK-3β RNA interference. The levels of hydrogen peroxide (H2O2), superoxide anion (O2.-), and hydroxy radical (·OH) as well as the activity of superoxide dismutase (SOD) were increased, while the activities of catalase (CAT) and glutathione peroxidase (GSH-PX) were decreased in the spleen and ZF4 cells of zebrafish by Li+ treatments. In addition, GSK-3β RNA interference increased ROS levels and decreased the activities of CAT and GSH-PX in the spleen. The fluorescence intensity of ROS was increased but the mitochondrial membrane potential (MMP) was decreased by Li+ treatments in ZF4 cells labeled with 2',7'-dichlorofluorescein diacetate (DCFH-DA) and Rhodamine-123, respectively. The results of present study indicated that the inhibition of GSK-3β promoted the ROS production via β-catenin/C/EBPα signaling in the spleen of zebrafish, and the balance between ROS and antioxidants could be destroyed by the GSK-3β/β-catenin/C/EBPα signaling. The results may be a valuable contribution to understanding the modulatory mechanism of GSK-3β/β-catenin/C/EBPα signaling on the antioxidant system in fish species.