Hydroxytyrosol bioaccessibility and absorption by the intestinal cells have been studied using an in vitro digestion model and Caco-2 TC7 monolayers cells in culture, in the presence or absence of β-cyclodextrin and foods. Hydroxytyrosol was either provided as a pure standard or in an alperujo powder. The presence of foods significantly decreased hydroxytyrosol bioaccessibility and absorption (-20% and -10%, respectively), while β-cyclodextrin had no effect. Moreover, the presence of other compounds from alperujo in the intestine compartment reduced hydroxytyrosol absorption by Caco-2 cells compared to pure standard (-60%). The final bioavailability of hydroxytyrosol, defined as its quantity at the basolateral side of cultured cell monolayers compared to the initial amount in the test meal, was 6.9±0.4%, 31.1±1.1% and 40.9±1.5% when hydroxytyrosol was from al perujo, or a standard administered with or without food, respectively. Our results show that conversely to foods, β-cyclodextrin does not alter hydroxytyrosolbioavailability.