The effect of copper(II) ions (Cu(+2)) on the structure of β-lactoglobulin (β-lg) was investigated spectroscopically using UV-visible, fluorescence and circular dichroism (CD) and calorimetrically using isothermal titration calorimetry (ITC), at different temperatures. Results of the UV-visible studies showed that adding Cu(+2) to β-lg solution caused increasing turbidity, indicative of protein aggregation. It was noticeable that the rate of increasing turbidity was directly proportional to increasing temperature. The far-UV CD studies displayed that the Cu(+2) cannot induce any significant changes in the secondary structures of β-lg at different temperatures. Also, the ITC data indicated that the binding process of Cu(+2) to β-lg is mainly entropically driven. The results highlight that copper ions cause the tertiary structure of β-lg to change and induce a slightly open structure leading to the formation of supramolecular aggregates in β-lg which may result in the reduced allergenicity of β-lg and its increased use in industrial applications.