Far-red fluorescent proteins (FPs) enable deep-tissue in vivo imaging. Combining FPs with large and small Stokes shifts enables single-excitation/dual-emission multicolor applications. Using a quantum mechanics/molecular mechanics (QM/MM) scheme, we carried out a series of simulations to identify the origin of an extended Stokes shift (0.2 eV) observed in mPlum, one of the most far-red-shifted FPs. We demonstrated that the red shift of emission is largely due to the excited-state relaxation of the chromophore itself. Rigid protein environment suppresses the relaxation; however, if the hydrogenbond network around the chromophore is sufficiently flexible, it can rearrange upon electronic excitation, allowing the chromophore to relax. The reorganization of the hydrogen-bond network is driven by changes in bonding and charge distributions of the chromophore in the excited state. The ILE65 and GLU16 residues play the most important role. The MD simulations reveal two ground-state populations with the direct (Chro-LLE65 center dot center dot center dot GLU16) and water-mediated (Chro-ILE65 center dot center dot center dot Wat321 center dot center dot center dot GLU16) hydrogen-bond patterns. In the excited state, both populations relax to a single emitting state with the water-mediated (Chro-ILE65 center dot center dot center dot Wat321 center dot center dot center dot GLU16) hydrogen-bond pattern, which provides a better match for the excited-state charge distribution (the acylimine's oxygen has a larger negative charge in S-1 than in S-0). The extended Stokes shift arises due to the conversion of the direct hydrogen-bond pattern to the water-mediated one accompanied by large structural relaxation of the electronically excited chromophore. This conclusion is supported by calculations for the GLU16LEU mutant, which has only one hydrogenbond pattern. Consequently, no interconversion is possible, and the computed Stokes shift is small, in agreement with the experiment. Our theoretical findings provide support to a recent study of the Stokes shifts in mPlum and its mutants.