High temperature reduces anthocyanin accumulation in various horticultural plants. However, the molecular mechanisms underlying the high-temperature-induced reduction of anthocyanin in grape (Vitis vinifera) remain poorly understood. In this study, VvMYB44-1 was identified as a transcriptional repressor of anthocyanin biosynthesis in grape berries, and its gene expression was strongly induced by high-temperature treatment. Overexpression of VvMYB44-1 inhibited anthocyanin accumulation in both grape berries and tobacco (Nicotiana tabacum) by repressing the transcription of the anthocyanin biosynthesis genes dihydroflavonol-4-reductase (VvDFR) and UDP-glucose flavonoid-3-O-glucosyltransferase (VvUFGT). Furthermore, the interaction between VvMYB44-1 and VvWDR2 competitively inhibited the formation of the MYB-bHLH-WD40 (MBW) activation complex and weakened the transcriptional activity of the complex, thereby decreasing anthocyanin accumulation. Additionally, VvMYB44-1 facilitated cytokinin (CK) accumulation by upregulating the expression of the CK synthesis gene lonely guy 8 (VvLOG8) and inhibiting the CK degradation gene CK oxidase 4(VvCKX4), thus contributing to CK-mediated anthocyanin inhibition in grape berries. Moreover, the inhibitory effect of VvMYB44-1 on anthocyanin biosynthesis and its downstream target genes was weakened with the deletion of the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif, indicating that the EAR motif is indispensable for the inhibitory effect of VvMYB44-1 on anthocyanin biosynthesis in grapes. These results provide insights into the regulatory network of VvMYB44-1 in high-temperature-mediated anthocyanin biosynthesis in grapes.