Rhodopsin is the G‐protein‐coupled receptor (GPCR) responsible for scotopic vision in the retina. Up to this point, the role of water in the activation of GPCRs has remained largely unknown. Recently, however, nanosecond molecular dynamics simulations have revealed an influx of bulk water into rhodopsin during activation . Utilizing rhodopsin as a model GPCR, we tested the hypothesis that rhodopsin activation is hydration mediated using osmotic stress techniques. We subjected rhodopsin within its native lipid membranes to varying osmotic pressures induced by different‐sized polyethylene glycol polymers. UV‐Visible spectroscopy of the photoactivated rhodopsin system reveals the fraction of protein in the active metarhodopsin‐II (MII) conformation, the receptor state capable of activating the G‐protein. We discovered high‐molecular weight osmolytes uniformly favored the closed, inactive metarhodopsin‐I conformation by dehydration of the protein interior. By contrast, small osmolytes penetrated into the transducin binding cleft and stabilized the active MII conformation until a quantifiable saturation point. A universal osmotic response occurred in the limit of increasing osmolyte size and maximal polymer exclusion from rhodopsin. By measuring the thermodynamic dependence of the metarhodopsin equilibrium on osmotic pressure, we determined that rhodopsin activation is coupled to a bulk influx of 80–100 water molecules into the protein core with a substantial increase in compressibility. We propose a new model for the functional role of water in GPCR signal transduction, in which a wet‐dry cycling mechanism amplifies the activation of G‐proteins. Our results necessitate a new understanding of GPCR activation, in which the influx of water plays a critical role in establishing the active receptor conformation.