Carotenoids are essential components of the photosynthetic apparatus and precursors of plant hormones, such as strigolactones (SLs). SLs are involved in various aspects of plant development and stress-response processes, including the establishment of root and shoot architecture. SL biosynthesis begins with the reversible isomerization of all-trans-carotene into 9-cis-β-carotene, catalysed by DWARF27 β-carotene isomerase (D27). Sequence comparisons have revealed the presence of D27-related proteins in photosynthetic eukaryotes and cyanobacteria lacking SLs. To gain insight into the evolution of SL biosynthesis, we characterized the activity of a cyanobacterial D27 protein (CaD27) from Cyanobacterim aponinum, using carotenoid-accumulating Escherichia coli cells and in vitro enzymatic assays. Our results demonstrate that CaD27 is an all-trans/cis and cis/cis-β-carotene isomerase, with a cis/cis conversion preference. CaD27 catalysed 13-cis/15-cis-, all-trans/9-cis-β-carotene, and neurosporene isomerization. Compared with plant enzymes, it exhibited a lower 9-cis-/all-trans-β-carotene conversion ratio. A comprehensive genome survey revealed the presence of D27 as a single-copy gene in the genomes of 20 out of 200 cyanobacteria species analysed. Phylogenetic and enzymatic analysis of CaD27 indicated that cyanobacterial D27 genes form a single orthologous group, which is considered an ancestral type of those found in photosynthetic eukaryotes. This article is part of the theme issue 'The evolution of plant metabolism'.