This study demonstrates gel-to-fluid transition temperatures of polydiacetylene bilayer vesicles could play important roles in their colorimetric transition temperatures. We prepared five types of polydiaceylene vesicles with 10,12-pentacosadiynoic acid (PCDA) and cholesterol (0-40 mol % of total content). From temperature-dependent observations of the optical signals (colors and UV-vis spectra), the blue-to-red colorimetric transition temperatures of polydiacetylene vesicles were decreased with the cholesterol contents. A further study with microcalorimetry and dynamic light scattering revealed that the polydiacetylene vesicles first underwent gel-to-fluid transitions, which were followed by event(s) responsible for the colorimetric transitions. Energies required for each event were quantified from analysis of the peaks in the microcalorimetry thermograms. The inclusion of cholesterol in the vesicles decreased both the gel-to-fluid and the colorimetric transition temperatures, suggesting that the colorimetric transition of the polydiacetylene vesicles was mediated by the former event although the event was not the direct reason for the color change.