© 2015 American Chemical Society. Rhodopsin, the visual pigment in the retina, is a Class A G protein-coupled receptor (GPCR) covalently bound to retinal chromophore. In dark conditions, retinal is in the cis-isomeric state, stabilizing the rhodopsin inactive state as an inverse agonist. After light absorption, retinal undergoes an isomerization photoreaction to trans-retinal, which includes a conformational change of the receptor to its active state. In the absence of retinal, the apoprotein opsin presents a low level of constitutive activity, which depends on pH, with higher propensity of activation at acidic pH. To examine the effect and the underlying mechanism that protonation may have on opsin activation, a number of MD simulations were run varying the number and identity of acidic residues selected for protonation. Results show that the combined protonation of D83, E113, and E247 is of special relevance for the induction of receptor activation. Subsequent conformational analysis of the MD trajectories provides a structural mechanistic insight into the opsin activation process. Furthermore, because protonation seems to be a determining step in the activation of other GPCRs, the methodology and rationale used herein can be extended to mechanistic studies of GPCRs in general.