The enzyme DWARF27 (D27) catalyzes the reversible isomerization of all-trans- into 9-cis-β-carotene, initiating strigolactone (SL) biosynthesis. Genomes of higher plants encode two D27-homologs, D27-like1 and -like2, with unknown functions. Here, we investigated the enzymatic activity and biological function of the Arabidopsis D27-like1. In vitro enzymatic assays and expression in Synechocystis sp. PCC6803 revealed an unreported 13-cis/15-cis/9-cis- and a 9-cis/all-trans-β-carotene isomerization. Although disruption of AtD27-like1 did not cause SL deficiency phenotypes, overexpression of AtD27-like1 in the d27 mutant restored the more-branching phenotype, indicating a contribution of AtD27-like1 to SL biosynthesis. Accordingly, generated d27 d27like1 double mutants showed a more pronounced branching phenotype compared to d27. The contribution of AtD27-like1 to SL biosynthesis is likely a result of its formation of 9-cis-β-carotene that was present at higher levels in AtD27-like1 overexpressing lines. By contrast, AtD27-like1 expression correlated negatively with the content of 9-cis-violaxanthin, a precursor of ABA, in shoots. Consistently, ABA levels were higher in shoots and also in dry seeds of the d27like1 and d27 d27like1 mutants. Transgenic lines expressing GUS driven by the AtD27LIKE1 promoter and transcript analysis of hormone-treated Arabidopsis seedlings revealed that AtD27LIKE1 is expressed in different tissues and affects ABA and auxin. Taken together, our work reports a cis/cis-β-carotene isomerase that affects the content of both cis-carotenoid-derived plant hormones, ABA and SLs.