The stabilities of Ca2+-regulated ctenophore and coelenterate apo-photoproteins, apo-mnemiopsin (apo-Mne) and apo-aequorin (apo-Aeq), respectively, were compared biochemically, biophysically, and structurally. Despite high degrees of structural and functional conservation, drastic variations in stability and structural dynamics were found between the two proteins. Irreversible thermoinactivation experiments were performed upon incubation of apo-photoproteins at representative temperatures. The inactivation rate constants (kinact) at 50 °C were determined to be 0.001 and 0.004 min-1 for apo-Mne and apo-Aeq, respectively. Detailed analysis of the inactivation process suggests that the higher thermostability of apo-Mne is due to the higher activation energy (Ea) and subsequently higher values of ΔH* and ΔG* at a given temperature. According to molecular dynamics simulation studies, the higher hydrogen bond, electrostatic, and van der Waals energies in apo-Mne can validate the relationship between the thermal adaptation of apo-Mne and the energy barrier for the inactivation process. Our results show that favorable residues for protein thermostability such as hydrophobic, charged, and adopted α-helical structure residues are more frequent in the apo-Mne structure. Although the effect of acrylamide on fluorescence quenching suggests that the local flexibility in regions around Trp and Tyr residues of apo-Aeq is higher than that of apo-Mne, which results in it having a better ability to penetrate acrylamide molecules, the root-mean-square fluctuation of helix A in apo-Mne is higher than that in apo-Aeq. It seems that the greater flexibility of apo-Mne in these regions may be considered as a determining factor, affecting the thermal stability of apo-Mne through a balance between structural rigidity and flexibility.