The variations of the barrier heights in the decomposition of the firefly dioxetanone and its analogues with the electrostatic field produced by the active site amino acid residues in the firefly luciferase are examined by a density functional theory study of the high-energy intermediates of the three luciferins. The positive electric field along the long-axis direction of the luciferins lowers the activation energy and acts as an electrostatic catalyst in the thermolysis process. The calculated barrier heights for the firefly dioxetanone and its analogues surrounded by the firefly Photinus pyralis luciferase show that the energy barrier of the firefly dioxetanone is lowered by the luciferase but is raised for the other analogue. Thus, the thermolysis rate is enhanced for the natural D-luciferin and reduced for the other analogue by the firefly luciferase, which elucidates why the luciferase acts as a catalyst for the natural D-luciferin but makes some luciferins emit weaker light signals.