Background <p>Long-term agricultural practices have demonstrated that rational combinations of tillage practices and nitrogen fertilization can improve soil physical structure, enhance nitrogen use efficiency, increase crop yield, and sustain the sustainability of agricultural ecosystems. However, research on the molecular and metabolic mechanisms regulating crop growth under the interaction of tillage and nitrogen management remains limited. Based on a 20-year-long field experiment conducted at the Loess Plateau Experimental Station, this study established a two-factor interactive experiment involving two tillage modes (no-tillage, NT; conventional tillage, CT) and two nitrogen levels (no nitrogen application, CK; nitrogen fertilization, N). By integrating transcriptomic and metabolomic analyses, we systematically explored the phenotypic, physiological, and molecular regulatory mechanisms underlying the responses of winter wheat roots to different tillage practices combined with nitrogen fertilization.</p> Results <p>Phenotypic and physiological-biochemical analyses showed that the combined treatment of no-tillage and nitrogen fertilization (NT_N) significantly improved root morphological traits of winter wheat, including root length (RL), root volume (RV), and root-to-shoot ratio (RS), relative to conventional tillage. In addition, the NT_N treatment increased the activities of nitrogen metabolism-related enzymes such as nitrate reductase (NR) and glutamine synthetase (GS), while optimizing the root antioxidant system and maintaining cell membrane stability. Transcriptomic analysis identified a total of 11,675 differentially expressed genes (DEGs), which were mainly enriched in pathways of plant-pathogen interaction, plant hormone signal transduction, and the MAPK signaling pathway. Metabolomic analysis detected 728 metabolites belonging to 19 categories, dominated by terpenoids, lipids, and flavonoids. Differential metabolites were significantly enriched in amino acid metabolism, glycerophospholipid metabolism, and secondary metabolite biosynthesis. A combined transcriptomic and metabolomic analysis revealed that no-till cultivation combined with nitrogen application promotes root morphogenesis by activating the tryptophan metabolism–auxin signaling pathway, while also improving cell membrane stability by upregulating key genes involved in glycerophospholipid metabolism. Furthermore, this treatment synergistically regulates plant hormone signaling networks, ultimately achieving the dual effects of promoting growth and enhancing stress resistance.</p> Conclusion <p>This study clarifies the regulatory effects of tillage practices combined with nitrogen fertilization on root growth, gene expression, and metabolic reprogramming in winter wheat. No-tillage integrated with nitrogen fertilization is an optimal green and high-efficiency cultivation pattern for winter wheat in dry farming areas of Northwest China, which provides a theoretical basis for precise tillage and nitrogen management in dryland wheat fields.</p>