G-protein–coupled receptors (GPCRs) are membrane proteins that transduce information across lipid bilayers. Their dynamic conformational ensembles encompassing various inactive and active states can be biased by the lipids and surrounding aqueous environments. By using different polyethylene glycol (PEG) solutions, we explored the effect of osmotic pressure and lipid bilayer composition on the metarhodopsin equilibrium of the archetypical GPCR rhodopsin in native membranes and POPC recombinant membranes. Our results show a flood of ∼80 water molecules into the rhodopsin interior during photoactivation, forming a solvent-swollen Meta-II active state [1]. Under applied osmotic pressure, the overall equilibrium generally shifted to Meta-I. Dehydrating conditions favor Meta-I through an efflux of water from the protein interior, while increasing bilayer thickness and the monolayer spontaneous curvature favor Meta-II. The osmotic effect on the protein is more significant than the effect of the lipid bilayer. However, small osmolytes favored the Meta-II state at lower concentrations, because they can penetrate the protein core giving a lower excluded volume, decreasing the osmotic effect on the protein and favoring the Meta-II state. Furthermore, the metarhodopsin equilibrium was shifted towards the Meta-I state in POPC recombinant membranes compared to the native membrane environment. 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. 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. Our results delineate the crucial role of soft matter in regulating the metarhodopsin equilibrium in a membrane environment. [1] U.