Abstract: A controlled water-level simulation experiment was conducted to investigate the physiological and metabolic responses of Cupressus gigantea W. C. Cheng & L. K. Fu seedlings to flooding stress in Xizang. Five flooding intensities (0%, 25%, 50%, 75%, and 100%) and three flooding durations (0, 4, and 6 months) were established to explore changes in osmotic regulation, antioxidant enzyme activity, and endogenous hormone levels. Results showed that roots exhibited substantially greater sensitivity to flooding stress than leaves. After 4 months of flooding, malondialdehyde (MDA) content decreased by 21% in leaves and by 78% in roots compared with the 0-month treatment. Abscisic acid (ABA) levels in roots showed no significant difference under 25% and 100% flooding compared with the control (0%), but declined markedly under prolonged flooding (6 months), suggesting that exposure duration exerted a stronger influence on physiological indices than flooding intensity. In addition, proline content in roots increased by 2% and 66% under 25% and 50% flooding after 4 months, respectively, but decreased significantly by 86% and 92% after 6 months relative to the unflooded condition. These findings suggest that flooding durations of 4–6 months represent a physiological threshold for Cupressus gigantea seedlings, beyond which stress tolerance and metabolic homeostasis are significantly impaired.