The discovery of the light-gated ion channel channelrhodopsin (ChR) set the stage for the novel field of optogenetics, where cellular processes are controlled by light. However, the underlying molecular mechanism of light-induced cation permeation in ChR2 remains unknown. Here, we have traced the structural changes of ChR2 by time-resolved FTIR spectroscopy, complemented by functional electrophysiological measurements. We have resolved the vibrational changes associated with the open states of the channel (P₂³⁹⁰ and P₃⁵²⁰) and characterized several proton transfer events. Analysis of the amide I vibrations suggests a transient increase in hydration of transmembrane α-helices with a t_1/2 = 60 μs, which tallies with the onset of cation permeation. Aspartate 253 accepts the proton released by the Schiff base (t_1/2 = 10 μs), with the latter being reprotonated by aspartic acid 156 (t_1/2 = 2 ms). The internal proton acceptor and donor groups, corresponding to D212 and D115 in bacteriorhodopsin, are clearly different from other microbial rhodopsins, indicating that their spatial position in the protein was relocated during evolution. Previous conclusions on the involvement of glutamic acid 90 in channel opening are ruled out by demonstrating that E90 deprotonates exclusively in the nonconductive P₄⁴⁸⁰ state. Our results merge into a mechanistic proposal that relates the observed proton transfer reactions and the protein conformational changes to the gating of the cation channel. Significance It was always a dream to control cells and living animals by light. Discovery of channelrhodopsin turned the dream into reality because this light-activated cation channel is able to elicit action potentials with unprecedented spatial and temporal resolution. To unravel the underlying molecular mechanism, we have applied time-resolved IR spectroscopy, and we suggest how the observed proton transfer and the protein conformational changes lead to opening of the cation channel. Our results will not only contribute to the rational design of channelrhodopsin variants with improved properties, but also help to decipher the temporal sequence in the gating of ion channels.