Pretreatment strategies are used to enhance crop growth and yield in sandy soils. This study aims to investigate the effects of glycine betaine (GB) as a grain priming agent on growth parameters, yield components, and metabolic changes in barley (Hordeum vulgare L.) at various concentrations: 0.0, 5, 10, and 20 mM. The application of glycine betaine resulted in a significant enhancement in the growth and yield of barley. Conspicuously, a concentration of 10 mM GB significantly increased the activity of antioxidant enzymes. Furthermore, chlorophyll content, total soluble sugars, phenolic compounds, total flavonoids, and amino acids were markedly elevated at 20 mM GB, while levels of proline and hydrogen peroxide (H₂O₂) were reduced. Additionally, the protein profile exhibited GB-responsive patterns that varied with concentration. The enhanced antioxidant capacity, modified protein profiles, and biochemical constituents of barley underscore the effectiveness of osmo-priming with glycine betaine in promoting growth, metabolic activities, and yield parameters in barley plants cultivated in sandy soil.