G-protein-coupled receptors (GPCRs) are responsible for transducing signals across lipid membranes in cells and are the largest family of targets (∼40%) for currently approved drugs. They exist as dynamic conformational ensembles with multiple inactive and active conformational substates described by an energy landscape model. We investigated ways in which the receptor hydration level and lipid bilayer composition influence the activation of the archetypical GPCR rhodopsin by quantifying the shift in the metarhodopsin equilibrium in native and POPC recombinant membranes by different polyethylene glycol osmolyte solutions. Our results show a flood of ∼90 water molecules into the rhodopsin interior during photoactivation, forming a solvent-swollen Meta-II active state. Dehydrating conditions favored inactive Meta-I through the efflux of water from the protein interior, while active Meta-II is favored by increasing bilayer thickness and the monolayer spontaneous curvature. The osmotic effect on the protein is more significant than the effect of the lipid bilayer, and hence the overall equilibrium was generally shifted to Meta-I. By contrast, small osmolytes can penetrate the protein core giving a lower excluded volume and stabilizing the Meta-II state. The metarhodopsin equilibrium was furthur shifted towards the Meta-I state in POPC recombinant membranes compared to the native lipid membrane environment. The POPC lipid membrane has zero-spontaneous curvature that shifts the equilibrium towards the more compact, inactive Meta-I state. By contrast, the native lipid membrane environment has a negative spontaneous curvature that favors the more expanded state of Meta-II. Analysis of transducin C-terminal peptide-binding isotherms revealed that the binding affinity is significantly decreased when the lipid environment is changed from the native lipids to POPC lipids. Our results delineate the crucial role of soft matter in regulating the activation process of GPCRs in a membrane lipid environment.