Abstract: In this study, the ‘Donghong’ and ‘Jinyu’ Actinidia varieties were treated with 20 mg/L CPPU at different periods of fruit development, with water used as a control. To explore the optimal period of CPPU treatment, differences in fruit quality, such as fruit weight, soluble solids content, sugar content, acid content, Vc content, and anthocyanin content, were estimated in different experimental groups. Real-time fluorescence polymerase chain reaction (RT-qPCR) was used to analyze the expression levels of anthocyanin-related genes in the CPPU-treated and control groups. Results showed that 14 d after flowering (14 DAF) was the best period for CPPU treatment in ‘Donghong’. Fruit weight and total sugar content in ‘Donghong’ increased by 24% and 38%, respectively, with CPPU treatment at 14 d. Correlation analysis showed that fruit weight was positively correlated with anthocyanin content. The ‘Jinyu’ fruit endocarp did not show significant changes after CPPU treatment. However, in the ‘Donghong’ fruit, anthocyanin content increased significantly and the endocarp became much redder after CPPU treatment. RT-qPCR analysis showed that the expression levels of the AcF3GT, AcF3H, AcLDOX, AcMYB10, and AcMYB110 genes were up-regulated after CPPU treatment, which promoted anthocyanin accumulation. Thus, CPPU treatment improved fruit quality in ‘Donghong’ and ‘Jinyu’ kiwifruit, and treatment at 14 d after flowering was optimal for ‘Donghong’. The expression levels of anthocyanin-related genes (AcF3GT, AcF3H, AcLDOX, AcMYB10, and AcMYB110) increased with CPPU treatment, which led to an increase in anthocyanin content and enhanced red flesh color in ‘Donghong’, thus providing a basis for studying the color mechanism and cytokinin regulation in Actinidia.