Bixin and crocin are natural apocarotenoids utilized as food colorants and additives in food industries worldwide. For safety assessment, it is necessary to understand the biological interaction of food colorants. In our present study, we report the interaction of two apocarotenoids with bovine serum albumin (BSA) at physiological pH using spectroscopic techniques and in silico tools. The binding constant and the mode of binding sites have been studied. The enthalpic and entropic contribution to the intermolecular binding event was analyzed and it was found that the contribution of hydrogen bonding and hydrophobic interactions was dominant. The adverse temperature dependence in the unusual static quenching is found to b. reasonable consequence of the large activation energy requirement in the binding process, which is required to overcome the fundamental block and i. direct result of the unique microstructure of the binding sites. To confirm the experimental analysis, we investigated the binding patterns using different in silico tools. combination of molecular docking, molecular dynamics, and toxicity analysis was performed, and the obtained results revealed that both the apocarotenoids had high binding affinity wit. binding energy of −5.44 and −5.93 kcal/mol for bixin and crocin, respectively, with no toxic effects and are in accordance with our experimental analysis. The results directly revealed the flexibility of the protein toward bixin and crocin which ha. great impact on the interaction. Thus bixin and crocin can guardedly be used as food colorants in food industries. © 2017 Informa UK Limited, trading as Taylor & Francis Group.