Brazilein extract from sappan wood (Caesalpinia sappan L.) has potential for use as natural food colorant since it has no unique flavor and taste. Although brazilein has long been applied in several traditional foods and beverages, information on its stability, which is of importance for practical application, is still limited. In this work, brazilein was isolated from sappan wood; its purity was confirmed by nuclear magnetic resonance spectroscopy. Relations between molecular structures and color as well as thermal stabilities of brazilein in aqueous solutions at pH 3, 7 and 9 were for the first time investigated. At the lowest pH, zero net-charge structure of brazilein, which exhibited yellow color, was predominantly found. The deprotonated and fully deprotonated structures of brazilein, which exhibited orange and red colors, respectively, were found when pH of the aqueous solutions increased. The forms of brazilein existing at the higher pH suffered extensive degradation upon heating, while the form existing at the lowest pH possessed higher stability. Heat-induced deprotonation and degradation were confirmed by UV-visible and Fourier-transform infrared spectra as well as losses of brazilein content.