13C-isotope feeding experiments demonstrate that the apocarotenoid 9-cis-β-apo-10'-carotenal is the precursor of several strigolactones in rice, providing a direct, in planta evidence for its role in strigolactone biosynthesis. Strigolactones (SLs) are plant hormone that regulates plant architecture and mediates rhizospheric communications. Previous in vitro studies using heterogously produced enzymes unraveled the conversion of all-trans-β-carotene via the intermediate 9-cis-β-apo-10'-carotenal into the SL precursor carlactone. However, a direct evidence for the formation of SLs from 9-cis-β-apo-10'-carotenal is still missing. To provide this evidence, we supplied rice seedlings with 13C-labeled 9-cis-β-apo-10'-carotenal and analyzed their SLs by LC-MS. Our results show that 9-cis-β-apo-10'-carotenal is the SL precursor in planta and reveal, for the first time, the application of labeled long-chain apocarotenoids as a promising approach to investigate apocarotenoid metabolism and the genesis of carotenoid-derived growth regulators and signaling molecules.