Iron overload in β-thalassemia promotes the accumulation of redox-active iron species such as non-transferrin-bound iron (NTBI) and labile plasma iron (LPI), which cause oxidative stress and tissue damage. Brazilin, a natural phenolic compound from Caesalpinia sappan L., has antioxidant and iron-chelating properties. However, its role in protection against iron-related toxicity remains unclear. In this study, we investigated the effects of brazilin on iron overload and oxidative stress in iron-loaded β-thalassemia knockout (BKO) mice. Iron overload was induced with a 0.2% TMH-ferrocene diet, and mice were treated with deferiprone (DFP 50 mg/kg body weight) or brazilin (50 and 100 mg/kg body weight). Iron parameters, oxidative stress markers, iron-regulatory gene expression, and histopathological changes were evaluated. Iron-loaded BKO mice showed increased levels of NTBI, LPI, and non-heme iron in red blood cells, tissue iron accumulation, lipid peroxidation, and altered hepcidin ( HAMP ) and transferrin receptor ( TfR ) expression. Brazilin substantially reduced NTBI, LPI, hepatic iron accumulation, and malondialdehyde (MDA) levels, and increased glutathione levels in the plasma and liver. It also modulated HAMP and TfR expression, and histologically reduced hepatic iron deposition, although the reduction in cardiac iron levels was modest. These findings demonstrate that brazilin attenuates iron overload and associated oxidative stress in β-thalassemic mice, consistent with its iron-chelating and antioxidant properties, and support its potential as an adjunctive approach for managing iron overload in β-thalassemia.