Osmoregulation is a vital physiological function for fish, as it helps maintain a stable intracellular concentration of ions in environments of variable salinities. We focused on a primarily freshwater species, the European whitefish (Coregonus lavaretus), to investigate the molecular mechanisms underlying salinity tolerance and examine whether these mechanisms differ between genetically similar populations that spawn in freshwater vs. brackishwater environments. A common garden experiment involving 27 families in two populations and five salinity treatments together with a large-scale, high-resolution mass spectrometry experiment that quantified 1500 proteins was conducted to assess phenotypic and proteomic responses during early development, from fertilization until hatching, in the studied populations. The populations displayed drastically different phenotypic and proteomic responses to salinity. Freshwater-spawning whitefish showed a significantly higher mortality rate in higher salinity treatments. Calcium, an ion involved in osmotic stress sensing, had a central role in the observed proteomic responses. Brackishwater-spawning fish were capable of viable osmoregulation, which was modulated by cortisol, an important seawater-adaptation hormone in teleost fish. Several proteins were identified to play key roles in osmoregulation, most importantly a highly conserved cytokine, tumour necrosis factor, whereas calcium receptor activities were associated with salinity adaptation. These results imply that individuals from these populations are most likely adapted to their local environments, even though the baseline level of genetic divergence between them is low (FST = 0.049). They also provide clues for choosing candidate loci for studying the molecular basis of salinity adaptation in other species. Further, our approach provides an example of how proteomic methods can be successfully used to obtain novel insights into the molecular mechanisms behind adaptation in non-model organism.