RsWRKY44 transcription factor, associated with anthocyanin biosynthesis in different radish cultivars, highly facilitates the activation of RsCHI and RsUFGT genes through its interaction with RsMYB1a, thereby promoting anthocyanin production. The regulation of anthocyanin biosynthesis in radish is primarily controlled by RsMYB1a and RsbHLH4, while the involvement of other factors in this process is not well understood. This study identified a WRKY transcription factor, RsWRKY44, as a key player in anthocyanin biosynthesis regulation. The expression of RsWRKY44 showed a strong correlation with anthocyanin content across different radish cultivars. RsWRKY44 was found to be expressed in the nuclei and exhibit transactivation activity. It was observed that only when RsWRKY44 was co-expressed with RsMYB1a, anthocyanin accumulation was induced in tobacco leaves, while RsWRKY44 alone did not. Additionally, RsWRKY44, along with RsMYB1a, activated the expression of tobacco endogenous anthocyanin biosynthesis regulatory genes NtAN1a and NtAN1b, as well as the structural genes NtCHS, NtCHI, NtDFR, NtF3H, NtANS, NtUFGT in transgenic tobacco. BiFC, FLC, and DLA assays confirmed the interaction between RsWRKY44 and RsMYB1a leading to the activation of radish genes RsCHI and RsUFGT, promoting anthocyanin biosynthesis. This study sheds light on a new molecular mechanism of RsWRKY44 involved in anthocyanin biosynthesis regulation in radish.