Nitration of tyrosine occurs under oxidative stress in vivo. The product, 3-nitrotyrosine (3NY), has a dramatically decreased quantum yield and can be used as a molecular ruler. In this study, femtosecond transient absorption spectroscopy and quantum calculations were implemented to elucidate the photoinduced relaxation processes of anionic 3NY in water. Upon 400 nm excitation into an excited electronic state with notable charge-transfer (CT) character, a barrierless nitro-twisting motion rapidly (<100 fs) guides the chromophore into an adjacent twisted intramolecular CT state, therein reaching a sloped S-1/S-0 conical intersection on the similar to 100 fs time scale. Once in the hot ground state, excess energy is further released through vibrational cooling with biexponential time constants of similar to 140 and 680 fs in water. Nitro back-twisting occurs on longer time scales (similar to 1.1 and 9 ps in water), returning the system to original ground state. Systematic evaluations of excited-state potential energies of anionic 3NY were performed by density functional theory (DFT) and time-dependent DFT calculations, showing that intersystem crossing (ISC) from the first singlet state (S-1) to the first or second triplet state (T-1 or T-2) is unlikely. Inclusion of an explicit water molecule in calculations leads to improved mapping of the excited-state energy ordering of the second singlet state (S-2) and T-2, further diminishing ISC probability from Si and favoring an ultrafast internal conversion to So. These results provide deep insights into the highly efficient nonradiative decay of anionic 3NY in aqueous solution, with nitro-site-specific information that can help infer the characterization and potential optogenetic control of 3NY in protein environment.