Background <p>Water content directly affects the dormancy progression and growth of grapevine (<i>Vitis vinifera</i> L.) winter buds. Insufficient water content in cane will disrupt winter bud dormancy, leading to physiological disorders in germination and flowering, which ultimately compromises yield and quality.</p> Methods and Results <p>To understand the regulatory mechanism of water content in cane on winter bud dormancy, a gradient of water treatments (T1 - T6, representing a decrease of 5% to 30% relative to the control) was applied to the canes of <i>Vitis vinifera</i> ‘Pinot Noir’ during dormancy. The forced single - node cuttings experiment and Kaplan - Meier analysis showed that the dormancy depth of winter buds deepened as the water content in cane decreased. The final germination percentages at T4, T5, and T6 respectively decreased by 20%, 50%, and 80% compared with the control. The ROS level revealed that this process was accompanied by ROS changes: the contents of H₂O₂ and O₂.⁻ increased as the water content in cane decreased. Concurrently, the activities of SOD and APX decreased, while the activities of POD and CAT increased. The transcriptomic analysis of CK and the treatments (T3, T5, T6) with significant differences in the final germination percentage showed that differentially expressed genes were significantly enriched in the “peroxidase activity” pathway. Notably, several peroxidase-related genes (e.g., <i>VvPRX10</i>) and transcription factors (e.g., bZIPs and AP2/ERFs) were identified. This indicates that antioxidant enzyme system, bZIPs, and AP2/ERFs play important roles in regulating winter bud dormancy under different cane water content condition .</p> Conclusions <p>This study reveals that the water content in cane regulates grapevine bud dormancy through the ROS homeostasis and the antioxidant enzyme system. The findings provide a scientific basis for improving the dormancy theory of grapevine and formulating precise water management strategies in viticulture.</p>