AbstractChannelrhodopsin (ChR) is a key protein of the optogenetic toolkit. C1C2, a functional chimeric protein of Chlamydomonas reinhardtii ChR1 and ChR2, is the only ChR whose crystal structure has been solved, and thus uniquely suitable for structure-based analysis. We report C1C2 photoreaction dynamics with ultrafast transient absorption and multi-pulse spectroscopy combined with target analysis and structure-based hybrid quantum mechanics/molecular mechanics calculations. Two relaxation pathways exist on the excited (S₁) state through two conical intersections CI₁ and CI₂, that are reached via clockwise and counter-clockwise rotations: (i) the C13=C14 isomerization path with 450 fs via CI₁ and (ii) a relaxation path to the initial ground state with 2.0 ps and 11 ps via CI₂, depending on the hydrogen-bonding network, hence indicating active-site structural heterogeneity. The presence of the additional conical intersection CI₂ rationalizes the relatively low quantum yield of photoisomerization (30 ± 3%), reported here. Furthermore, we show the photoreaction dynamics from picoseconds to seconds, characterizing the complete photocycle of C1C2.