Droughts represent one of the main challenges that climate change imposes on crop production. As a globally cultivated staple crop, wheat (Triticum aestivum L.) is prone to drought environments. Therefore, improvement in drought tolerance represents a growing concern to ensure food security, especially for wheat. In this perspective, the application of Phyto-phillic exogenous materials such as glycine-betaine (GB) has been attracting attention, particularly in stress-related studies. Since roots procure the water and nutrients for plants, any improvements in their response and capacity against drought stress could induce stress tolerance in plants. However, the knowledge about the changes in root architecture, defense mechanism, hormonal metabolism, and downstream signaling, in response to GB-mediated root priming, is still limited. Therefore, we designed the present study to investigate the role of GB-mediated root priming in improving the water stress tolerance in wheat (cv. Jimai-22) under in-vitro conditions. The roots of twelve days old wheat seedlings were treated with Hoagland’s solution (GB-0), 50 mM GB (GB-1), and 100 mM GB (GB-2) for 48 h and subjected to well-watered (WW) and water-stress (WS) conditions. The osmotic stress substantially impaired shoot/root growth, dry matter accumulation, and increased malondialdehyde (MDA) and hydrogen-peroxide (H2O2) production in the roots of wheat seedlings. However, GB-mediated root priming improved the redox homeostasis of wheat roots by boosting the activities of SOD and POD and triggering the significantly higher accumulation of abscisic acid (ABA) and salicylic acid (SA) in the roots of GB-primed plants. Consequently, it modified the root architecture system and improved plant growth, dry matter accumulation, and water-stress tolerance of wheat seedlings. Moreover, GB-mediated root priming increased root sensitivity to water stress and induced overexpression of stress-responsive genes involved in ABA metabolism (TaNECD1, TaABA’OH2), their downstream signal transduction (TaPP2C, TaSNRK2.8), and activation of different transcriptional factors (TabZIP60, TaAREB3, TaWRKY2, TaERF3, and TaMYB3) that are associated with plant metabolite accumulation and detoxification of ROS under water stress conditions. Overall, our results demonstrated that GB-priming improved the physiological and biochemical attributes of wheat plants under WS conditions by improving the drought perception capacity of wheat roots, ultimately enhancing the water stress tolerance. Thus, the GB-priming of roots could help to enhance the water-stress tolerance of economically important crops (i.e., wheat).