Lutein is a xanthophyll involved in the prevention of age-related macular degeneration. Its health effects obviously depend on its bioavailability, which can be modulated by the food matrix. Indeed, the matrix can either hold back its embedded micronutrients, or protect them from a possible degradation in the upper gastrointestinal tract. Thus, the purpose of this study was to determine the impact of the matrix of canned spinach on lutein bioavailability. In vitro digestions were first used to compare lutein bioaccessibility (percentage level of lutein released from the matrix to mixed micelles) when lutein was added to the test meal either as a pure molecule or in canned spinach. Then, mixed micelles isolated from these in vitro digestions were delivered to human intestinal Caco-2 TC7 cells to assess lutein uptake and efflux. To strengthen these results, an experiment was performed in piglets to verify in vivo the impact of the canned spinach matrix on lutein bioavailability, which was evaluated by comparison of the postprandial plasma lutein responses (0-12 h area under the curve of the plasma lutein concentrations) after test meals that contained either pure lutein or lutein in canned spinach. Our results showed that lutein bioaccessibility was significantly higher when lutein was digested in spinach as compared to in vitro digestion of the pure form (+35%, p<0.05). However, lutein uptake by Caco-2 cells was less effective when lutein was given in micelles containing its pure form compared to micelles isolated from spinach digestion (-55%, p<0.05). This second step contributed to decrease the favorable effect of spinach observed during in vitro digestions. These data were confirmed by the postprandial study in piglets where no significant difference was observed between postprandial lutein responses to the two test meals. Overall, our results reveal that lutein bioavailability does not significantly change whether it is ingested as a purified molecule or within its usual vegetable matrix. However, it is important to remind that spinach consumption remains interesting since it brings both large quantities of lutein as well as other micronutrients that likely display additional health effects besides lutein properties.