Abstract: Ascorbic acid (AsA) oxidation is a critical biological process involved in maintaining ascorbic acid homeostasis. However, its regulation by abscisic acid (ABA) and associated biological effects remain unknown. This study selected OsAAO4, an ascorbate oxidase gene in rice (Oryza sativa L.), to analyze its response to ABA and the phenotypic and physiological effects of its knockout under ABA treatment. Results showed that OsAAO4 contained abundant ABA response elements and reached peak expression levels in roots and young leaves after 9 h of ABA treatment, increasing 13.8-fold and 7.4-fold, respectively. Under normal conditions without ABA treatment, there were no significant differences in plant height and survival rate between the wild-type (‘Zhonghua 11’) and OsAAO4 knockout lines (KO-1 and KO-2). However, exposure to 10 μmol/L ABA resulted in significant reductions in plant height (38.34% in KO-1 and 25.72% in KO-2) and survival rate (21.91% in KO-1 and 5.48% in KO-2) compared to wild-type rice. Additionally, in young leaves, the knockout lines exhibited decreased ascorbic acid oxidase (AAO) activity (35.31% in KO-1 and 31.81% in KO-2) and dehydroascorbic acid (DHA) content (29.72% in KO-1 and 26.95% in KO-2), while AsA content increased (27.17% in KO-1 and 16.37% in KO-2). Transcriptomic analysis revealed that differentially expressed genes (DEGs) between KO-1 and wild-type rice under ABA treatment were significantly enriched in pathways involved in redox homeostasis and oxidative stress. These findings suggest that ascorbic acid oxidation mediated by OsAAO4 plays an important role in maintaining AsA homeostasis and ensuring the normal growth of rice seedlings under ABA treatment.