Since the food matrix determines β-carotene availability for intestinal absorption, food matrix effects on the bioaccessibility of β-carotene from two diets were investigated in vitro and compared with in vivo data. The "mixed diet" consisted of β-carotene-rich vegetables, and the "oil diet" contained β-carotene-low vegetables with supplemental β-carotene. The application of extrinsically labeled β-carotene was also investigated. The bioaccessibility of β-carotene was 28 μg/100 μg β-carotene from the mixed diet and 53 μg/100 μg β-carotene from the oil diet. This ratio of 1.9:1 was consistent with in vivo data, where the apparent absorption was 1.9-fold higher in the oil diet than in the mixed diet. The labeled β-carotene was not equally distributed over time. In conclusion, the food matrix effects on bioaccessibility of β-carotene could be measured using an in vitro model and were consistent with in vivo data. The application of extrinsically labeled β-carotene was not confirmed.