Temporal and spatial variations in the water status of walnut trees (Juglans regia L.) and the soil in which they were growing were traced by analyzing the differences in hydrogen isotopes during spring and summer in a 7-year-old walnut stand. Walnut root dynamics were measured in both dry and wet seasons. Walnut roots were mainly distributed in the upper soil (0–30 cm depth), with around 60% of the total root mass in upper soil layers and 40% in deep soil layers (30–80 cm depth). The upper soil layers contributed 68% of the total tree water requirement in the wet season, but only 47% in the dry season. In the wet season, total roots, living roots and new roots were all significantly more abundant than in the dry season. There were significant differences in pre-dawn branch percentage loss of hydraulic conductance (PLC), pre-dawn leaf water potential and transpiration between the dry and wet seasons. Water content in the upper soil layers remarkably influenced xylem water stable-hydrogen isotope (δD) values. Furthermore, there were linear relationships between the xylem water δD value and pre-dawn branch PLC, pre-dawn leaf water potential, transpiration rate and photosynthetic rate. In summary, J. regia was compelled to take a larger amount of water from the deep soil layers in the dry season, but this shift could not prevent water stress in the plant. The xylem water δD values could be used as an indicator to investigate the water stress of plants, besides probing profiles of soil water use.