AbstractIsocytosine, having important applications in antivirus and drug development, is among the building blocks of Hachimoji nucleic acids. In this report, we present an investigation of the excited state dynamics of isocytosine in both protic and aprotic solvents, which was conducted by a combination of methods including steady‐state spectroscopy, femtosecond broadband time‐resolved fluorescence, and transient absorption. These methods were coupled with density functional and time‐dependent density functional theory calculations. The results of our study provide the first direct evidence for a highly efficient nonradiative mechanism achieved through internal conversion from the ππ* state of the isocytosine keto‐N(3)H form occurring within subpicoseconds and picoseconds following photo‐excitation. Our study also unveils a crucial role of solvent, particularly solute–solvent hydrogen bonding, in determining the tautomeric composition and regulating the pathways and dynamics of the deactivation processes. The deactivation processes of isocytosine in the solvents examined are found to be distinct from those of cytosine and the case known for isocytosine in the gas phase mainly due to different tautomeric forms involved. Overall, our findings demonstrate the high photo‐stability of isocytosine in the solution and showcase the remarkable effect of covalent modification in altering the spectral character and excited state dynamics of nucleobases.