The aim of this paper is to report on the demineralization-induced changes in dentin autofluorescence. Confocal laser scanning microscopy (CLSM) images and fluorescence excitation and emission spectra in vitro demineralized root surfaces and sound controls on the same tooth roots were compared. When observed in CLSM images, demineralized dentin, excited at 488 nm, gave an increased emission at 529 nm compared to sound dentin. The difference in fluorescence decreased deeper into the root, as the sound dentin underneath the lesion was reached. In contrast, when using fluorescence spectrophotometry, excitation around 460 and 488 nm yielded a lower emission around 520 nm for demineralized dentin than for sound dentin, but in a more pronounced peak. From excitation spectra for emission around 520 nm, it could be seen that in demineralized dentin the contribution of excitation between 480 and 520 nm was more important than in sound dentin. The recorded fluorescence in CLSM images was not affected by demineralization-caused changes in scattering and absorption properties, due to the small measurement volume. Thus, the increased fluorescence for demineralized dentin implies an increased quantum yield. In fluorescence spectrophotometry, where the measurement volume is large, changes in scattering and absorption do have an influence on the fluorescence signal. Then, increased absorption by non-fluorescing chromophores and increased re-absorption around the emission wavelength may compensate for the increase in quantum yield and absorption around the excitation wavelength by fluorophores.