Physicochemical characteristics of traditional Tunisian butter (TTB) and thermal stability of the butter oil were studied. Thermal stability of samples was estimated by using the accelerated shelf life testing method. Effect of heating on some quality characteristics of traditional Tunisian butter oil (TTBO) was investigated and compared at different temperatures (60°C, 100°C and 130°C). Induction period of sample heated at 60°C was important compared to that at 100°C and 130°C. This result may indicate the sensitivity of TTBO to elevated temperature. Absorption at 232 and 270 nm, acidity and PV increased rapidly after reaching the oxidation induction time. The temperature had a significant effect on the formation of oxidation products in traditional butter oil (TTBO). Fatty acid composition of TTBO was also changed after heating. This may explain the observed modification on some quality characteristics of TTBO such as viscosity, hardness, cohesiveness and colour. As a consequence, heat treatment produced alterations in the oxidative status of the butter oil that could affect the shelf life.