Studies were conducted based on different intensities of the red-flesh color peaches to analyze the their formation mechanism and provide theoretical basis for efficient breeding of red peach varieties with different intensities. In this study, promoter activity of PpMYB10.1 was detected using GUS staining buffer, transcription repressors were captured through DNA-pull down assay, the function of candidate genes were determined by double luciferase and yeast two-hybrid assay. The results showed that expression of PpMYB10.1 and anthocyanin content of deep red, red and light red flesh peach decreased successively, indicating that the 483bp deletion and 5243bp insertion on PpMYB10.1 promoter have changed its promoter activity. Subsequently, we verified the 483bp deletion sequence and designed two promoter sequences, one of which contained the 483bp deletion sequence and 1609bp in length, and the other segment did not contain the 483bp deletion sequence and 1126bp in length. Their activities were identified by GUS staining. We found that activity of PpMYB10.1 promoter with 483bp sequence was weaker than that without the sequence. Later, we found that the activity of LUC at the injection site with ‘PpBL+PpNAC1+proMYB10.1(1126bp)’ was almost comparable to that of ‘PpBL+PpNAC1+proMYB10.1(1609bp)’, indicating that the presence or absence of the 483bp sequence had little effect on PpBL and PpNAC1 binding to the PpMYB10.1 promoter. Therefore, we believe that the 483bp sequence may bind to a transcriptional repressor, leading to the weakening of PpMYB10.1 initiation activity. Then, we designed the 483bp sequence as a probe, labeled with FGD fluorescein, and incubated with red peach fruit solid nucleoprotein. A total of 15 target proteins were identified, of which 2 (Prupe.2G302800 and Prupe.6G284800) showed inhibitory function according to functional annotation, and were used for subsequent functional verification. Dual luciferase assay was used to verify the transcriptional inhibitory activity of the two candidate genes. We found that the LUC activity of the injection site with ‘Prupe.2G302800 +proMYB10.1’ was stronger than that of the control‘GUS+proMYB10.1’ in tobacco leaves. However, with ‘Prupe.6G284800 +proMYB10.1’ injection site, LUC activity was almost comparable to that of control ‘GUS+proMYB10.1’. These results indicated that the two candidate genes had no inhibitory activity against the PpMYB10.1 promoter. Interestingly, the Prupe.2G302800 identified using the 483bp sequence showed strong interaction with PpBL, and inhibited its transcriptional activity to PpMYB10.1, which had a certain function in reducing the expression of PpMYB10.1. Although Prupe.2G302800 identified in this study is not the direct factor for lightening the red flesh of peach, it can play an important role in lightening the red flesh peach by inhabiting PpMYB10.1 transcription through interaction with PpBL.