Sweet potato (Ipomoea batatas) is a globally significant food and functional crop, with its purple-fleshed varieties drawing particular attention due to the high anthocyanin content. Anthocyanins not only give the tubers distinct color but also possess antioxidant, anti-inflammatory, and disease-preventive physiological functions, making them highly valuable in the development of health foods. However, the biosynthesis of anthocyanins is regulated by a multi-level network, and the role of bHLH transcription factors in sweet potatoes has not been fully elucidated. This study focused on the sweet potato gene Tai6.33665, aiming to clarify its molecular characteristics, subcellular localization pattern, and regulatory role in anthocyanin accumulation, providing a theoretical basis for molecular breeding of high-anthocyanin sweet potato varieties. Using purple-fleshed sweet potatoe (PFSP) and white-fleshed sweet potatoe (WFSP) as materials, the study analyzed the protein structure and evolutionary relationship of Tai6.33665 through bioinformatics, detected the gene expression pattern using qRT-PCR, constructed a Tai6.33665-GFP fusion vector for transient transformation in tobacco, determined the subcellular localization using laser confocal microscopy, and analyzed the correlation between gene expression and anthocyanin content and the miRNA interaction mechanism. The results showed that the protein encoded by Tai6.33665 had a typical bHLH domain and was highly homologous to anthocyanin synthesis regulatory factors. Its expression level was significantly negatively correlated with anthocyanin accumulation (P<0.05), and it was targeted and inhibited by ib-miR52. Subcellular localization indicated that the protein was located in the endoplasmic reticulum, suggesting that it might indirectly regulate metabolism through post-translational modification or the endoplasmic reticulum pathway. This study confirmed that Tai6.33665 acted as a negative regulator of anthocyanin biosynthesis in sweet potatoes, and its function depended on subcellular localization specificity and miRNA-mediated post-transcriptional regulatory networks. This discovery not only enriches the theoretical framework of plant secondary metabolism regulation but also provides a dual-track strategy for molecular breeding by targeting knockout of Tai6.33665 or overexpression of ib-miR52 using CRISPR/Cas9, the genetic limitations of anthocyanin accumulation could be overcome, and new high-anthocyanin sweet potato varieties could be developed. Future work would focus on elucidating the interaction mechanism between the gene and MYB/WD40 proteins, as well as the regulatory pathway of its transcriptional activity by endoplasmic reticulum localization.