Whether protein could adopt multiple conformations coexisting in solution is disputable. In a previous report, the conformation heterogeneity of apoazurin mutant M121L had been identified. The thermal unfolding of wild type apoazurin from Pseudomonas aeruginosa is re-investigated with differential scanning calorimetry (DSC) and circular dichroism (CD) methods. The results show that unfolding in the pH range from 4 to 9 is associated with two heat capacity maxima. The low temperature transitions are reversible at all pH conditions used, while the high temperature transitions are irreversible. The two unfolding transitions were analyzed by the two-interchangeable-conformation model with the fraction for the first transition (N1) from 64% at pH 4.0 to 55% at pH 9.0. Temperature induced unfolding monitored at 219 nm shows also two separate transitions. The ratio of the signal changes is consistent with the fractions obtained from the corresponding DSC measurements. These results provide further support for the hypothesis that at least two conformations of apoazurin coexists in solution.