The photoisomerization scheme of the cyanine-based 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiD) probe was investigated by means of molecular modeling techniques, accounting for differences between the potential energy surfaces in the ground and excited states. Starting from the trans conformation, the photoisomerization path to the cis conformation and its dependence on the acyl tail lengths of the probe were evaluated. Moreover, the ground-state conformational distribution was investigated and suitable topologies were built for the ground- and excited-state molecular dynamics (MD) calculations. A protocol for simulations in solvents and in liquid-disordered lipid bilayers was worked out. In a kinetic analysis, the decay of the excited singlet (S1) state via radiative and nonradiative decays and via dihedral twisting is discussed. The twisting of one of the dihedral angles in the S1 state is found to be faster than the direct decay rate, which explains the relatively low fluorescence quantum yield of the compound. The molecular dynamics simulations show that in lipid bilayers, the DiD probe with methyl groups as acyl tails from the headgroup brings the highest level of photoisomerization, while a compound with acyl tails of 18 carbon atoms does not isomerize at all.