Studying the cis–trans isomerization process in crocin (CR), one of the few water-soluble carotenoids extracted from saffron, is important to better understand the physiological role of cis-carotenoids in vivo and their potential as antioxidants in therapeutic applications. For that, cis–trans isomerization of both methanol- and water-dissolved CR was induced by light or thermally generated singlet oxygen (1O2). The kinetics of molecular concentrations were monitored by both high-performance liquid chromatography (HPLC) and non-destructive spectrophotometric methods. These last made it possible to simultaneously follow the cis–trans isomerization, the possible bleaching of compounds and the amount of thermally generated1O2. Our results were in accordance with a comprehensive model where the cis–trans isomerization occurs as relaxation from the triplet state of all-trans- or 13-cis-CR, whatever is the way to populate the CR triplet state, either by photon or1O2energy transfer. The process is much more (1.9 to 10-fold) efficient from cis to trans than vice versa. In H2O, a1O2-induced bleaching effect on the starting CR was not negligible. However, the CR “flip-flop” isomerization reaction could still occur, suggesting that this process can represent an efficient mechanism for quenching of reactive oxygen species (ROS) in vivo, with a limited need of carotenoid regeneration.