Alkaline stress with high pH levels could significantly influence plant growth and survival. The enzyme 9-cis-epoxycarotenoid dioxygenase (NCED) serves as a critical bottleneck in the biosynthesis of abscisic acid (ABA), making it essential for regulating stress tolerance. Here, we show that -overexpressing rice lines have increased ABA content by up to 50.90% and improved transcription levels of numerous genes involved in stress responses that significantly enhance seedling survival rates. Overexpression of increased the dry weight contents of the total chlorophyll, proline, soluble sugar, starch, and the activities of antioxidant enzymes of rice seedlings, while reducing the contents of O·, HO, and malondialdehyde under hydroponic alkaline stress conditions simulated by 10, 15, and 20 mmol L of NaCO. Additionally, the -overexpressing rice lines exhibited a notable increase in the expression of ; ABA response-related genes and ; ion homeostasis-related genes , , , and ; and ROS scavenging-related genes , , , , , and in rice seedling leaves. The results of these findings suggest that overexpression of upregulates endogenous ABA levels and the expression of stress response genes, which represents an innovative molecular approach for enhancing the alkaline tolerance of rice seedlings.