Consumers value highly the nutritional content and flavor of fresh fruits, which are influenced by endogenous plant hormones. However, the molecular mechanisms governing the hormonal regulation of essential nutrients such as ascorbic acid (AsA) in fruit are still unclear. This study investigates the regulation of AsA synthesis in kiwifruit by the transcription factor AcABI5a, which is involved in mediating the abscisic acid (ABA) signal. A negative correlation between AcABI5a expression and AsA levels across different developmental stages of kiwifruit was observed. Furthermore, AcABI5a was found to bind both the AcMYBS1 promoter, repressing its transcriptional activity, and its own promoter, fostering expression and maintaining active repression of AcMYBS1. AcMYBS1 activates the expression of AcGGP3, which encodes an enzymatic step in AsA biosynthesis that is highly regulated both transcriptionally and translationally. In-depth interaction studies utilizing yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), firefly luciferase complementation (NC-LUC), and pull-down assays unveiled that AcABI5a also physically interacts with AcMYBS1, further impeding its activation of AcGGP3. Results from knockout by gene editing and overexpression of AcABI5a support the role of AcABI5a in mediating the ABA inhibitory effect on AsA synthesis by repressing the expression of AcMYBS1 and thus AcGGP3. Overall, our findings highlight AcABI5a’s negative regulatory role in AsA synthesis by integrating ABA signaling during fruit development, providing new insights into the regulation of AsA synthesis by phytohormones.

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