Recently, a standardized in vitro digestion model (“INFOGEST method”) has been developed to evaluate the gastrointestinal fate of foods. Under fed state conditions, the calcium level used in this model is fixed and relatively low: 0.525 mM. In practice, the calcium concentration in the human gut depends on the nature of the food consumed and may vary from person-to-person. For this reason, we examined the impact of calcium concentration on the gastrointestinal fate of a model nutraceutical delivery system. The effect of calcium level (0.525-10 mM) on lipid digestion and β-carotene bioaccessibility in corn oil-in-water nanoemulsions was investigated using the INFOGEST method. At all calcium levels, the lipids were fully digested, but this could only be established by carrying out a back titration (to pH 9) at the end of the small intestine phase. Conversely, the bioaccessibility of β-carotene decreased with increasing calcium levels: from 65.5% at 0.525 mM Ca2+ to 23.7% at 10 mM Ca2+. This effect was attributed to the ability of the calcium ions to precipitate the β-carotene-loaded mixed micelles by forming insoluble calcium soaps. Our results show that the bioaccessibility of hydrophobic carotenoids measured using the INFOGEST method is highly dependent on the calcium levels employed, which may have important consequences for certain calcium-rich foods.