Background: The transcription regulator IbWD40 is known to be involved in anthocyanin biosynthesis in purple-flesh sweet potato (Ipomoea batatas). However, little is known about the upstream transcription regulators on the promoter of IbWD40. Methods: Yeast one-hybrid screening was performed on the storage roots of purple-fleshed sweet potato to identity upstream transcription regulators on the promoter of IbWD40. Luciferase reporter assays and Yeast one-hybrid assays were used to verify these upstream binding proteins interacted with the promoter. Real-time PCR was used to analyze the gene expression of upstream transcription regulators, transcription factors, and structural genes involved in anthocyanin biosynthesis in different root stages of purple-fleshed and white-fleshed sweet potato. Results: IbERF1, IbERF10, IbEBF2, IbPDC, IbPGP19, IbUR5GT, IbDRM, IbPPA and IbERF73 were identified as candidate binding proteins for the promoter of IbWD40. Furthermore, IbERF1, IbERF10 and IbERF73 were identified as upstream transcription regulators on the promoter of IbWD40 involved in anthocyanin biosynthesis. Conclusions: IbERF1, IbERF10 and IbERF73 were identified as transcription regulators on the promoter of IbWD40, which is involved in the regulation of anthocyanin biosynthesis in purple-fleshed sweet potato.