Steady-state emission and time-resolved techniques were employed to study the photoprotolytic processes D-luciferin undergoes in water-methanol mixtures over a wide range of molar fractions (chi(meOH)) of methanol. We found that in the concentration range of 0 < chi(MeOH) < 0.8 the rate constant of the excited-state proton transfer (ESPT) to the solvent decreases nearly exponentially with increasing chi(MeOH). At chi(MeOH) > 0.8 the proton transfer rate constant decreases with an even steeper slope. The kinetic isotope effect (KIE) maintains a constant value of 2.4 +/- 0.2 at all the mixture's compositions.