Chrysanthemum is an important ornamental crop worldwide. Some white-flowered chrysanthemum cultivars produce red ray florets under natural cultivation conditions, but little is known about how this occurs. We compared the expression of anthocyanin biosynthetic and transcription factor genes between white ray florets and those that turned red based on cultivation conditions to comprehend the underlying mechanism. Significant differences in the expression of were detected between the florets of different colors. generated two alternatively spliced transcripts, designated and . Compared with , encoded a truncated protein with only a partial MYB-interaction region and no other domains normally present in the full-length protein. Unlike the full-length form, the splicing variant protein CmbHLH2 localized to the cytoplasm and the nucleus and could not interact with CmMYB6. Additionally, CmbHLH2 failed to activate anthocyanin biosynthetic genes and induce pigment accumulation in transiently transfected tobacco leaves, whereas CmbHLH2 promoted both processes when simultaneously expressed with CmMYB6. Co-expressing CmbHLH2 and CmMYB6 also enhanced the promoter activities of and . Notably, the Arabidopsis mutant, which lacks red pigmentation in the leaves and seeds, could be complemented by the heterologous expression of CmbHLH2, which restored red pigmentation and resulted in red pigmentation in high anthocyanin and proanthocyanidin contents in the leaves and seeds, respectively, whereas expression of CmbHLH2 did not. Together, these results indicate that CmbHLH2 and CmMYB6 interaction plays a key role in the anthocyanin pigmentation changes of ray florets in chrysanthemum. Our findings highlight alternative splicing as a potential approach to modulate anthocyanin biosynthesis in specific tissues.