<jats:title>Summary</jats:title><jats:p>The synthesis of the acidic apo‐carotenoid neurosporaxanthin by the fungus <jats:italic>Fusarium fujikuroi</jats:italic> depends on four enzyme activities: phytoene synthase and carotene cyclase, encoded by the bifunctional gene <jats:italic>carRA</jats:italic>, a carotene desaturase, encoded by <jats:italic>carB</jats:italic>, and a postulated cleaving enzyme converting torulene (C<jats:sub>40</jats:sub>) into neurosporaxanthin (C<jats:sub>35</jats:sub>). Based on sequence homology to carotenoid oxygenases, we identified the novel fungal enzyme CarT. Sequencing of the <jats:italic>carT</jats:italic> allele in a torulene‐accumulating mutant of <jats:italic>F. fujikuroi</jats:italic> revealed a mutation affecting a highly conserved amino acid, and introduction of a heterologous <jats:italic>carT</jats:italic> gene in this mutant restored the ability to produce neurosporaxanthin, pointing to CarT as the enzyme responsible for torulene cleavage. Expression of <jats:italic>carT</jats:italic> in lycopene‐accumulating <jats:italic>E. coli</jats:italic> cells resulted in the formation of minor amounts of apo‐carotenoids, but no enzymatic activity was observed in β‐carotene‐accumulating cells, indicating a preference for acyclic or monocyclic carotenes. The purified CarT enzyme efficiently cleaved torulene <jats:italic>in vitro</jats:italic> to produce β‐apo‐4′‐carotenal, the aldehyde corresponding to the acidic neurosporaxanthin, and was also active on other monocyclic synthetic substrates. In agreement with its role in carotenoid biosynthesis, the <jats:italic>carT</jats:italic> transcript levels are induced by light and upregulated in carotenoid‐overproducing mutants, as already found for other <jats:italic>car</jats:italic> genes.