BRAFV600E comes as two main splicing variants. The well-studied ref isoform and the recently discovered X1 isoform are co-expressed in cancer cells and differ in terms of 3’UTR length and sequence, as well as C-term protein sequence. Here, we use a melanoma model in zebrafish to study the role played by each isoform in larval pigmentation, nevi formation, and their progression into melanoma tumours. We show that both BRAFV600E-ref and BRAFV600E-X1 proteins promote larval pigmentation and nevi formation, while melanoma-free survival curves performed in adult fish indicate that BRAFV600E-ref protein is a much stronger melanoma driver that BRAFV600E-X1 protein. Crucially, we also show that the presence of the 3’UTR suppresses the effect of ref protein. Our data highlight the necessity to undertake a systematic study of BRAFV600E isoforms, in order to uncover the full spectrum of their kinase-(in)dependent and coding-(in)dependent functions, hence to develop more informed strategies for therapeutic targeting.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-023-01064-w.