Abstract Flavin-binding light, oxygen, and voltage (LOV) domains are UVA/blue-light-sensing protein units that form a reversible flavin mononucleotide-cysteine adduct upon light induction. In their dark-adapted state, LOV domains exhibit the typical spectral features of fully oxidized riboflavin derivatives. A survey on the absorption spectra of various LOV domains revealed that the UVA spectral range is the most variable region (whereas the absorption band at 450 nm is virtually unchanged), showing essentially two distinct patterns found in plant phototropin LOV1 and LOV2 domains, respectively. In this work, we have identified a residue directly interacting with the isoalloxazine methyl group at C(7a) as the major UVA spectral tuner. In YtvA from Bacillus subtilis, this amino acid is threonine 30, and its mutation into apolar residues converts the LOV2-like spectrum of native YtvA into a LOV1-like pattern. Mutation T30A also accelerates the photocycle ca. 4-fold. Together with control mutations at different positions, our results experimentally confirm the previously calculated direction of the transition dipole moment for the UVA ππ* state and identify the mechanisms underlying spectral tuning in the LOV domains.