The amount of nutrients that can be released from food products (i.e., nutrient in vitro bioaccessibility) is often studied as it is a starting point for investigating nutrient bioavailability, an indicator for the nutritional value of food products. However, the importance of mastication as a particle size reduction technique is poorly understood and is often neglected during in vitro procedures determining bioaccessibility. Therefore, the aim of the present work was to study the effect of mechanical breakdown on the β-carotene bioaccessibility of carrot samples, having different textural/structural characteristics (as a result of thermal processing). In the first part of this study, the all-E-β-carotene bioaccessibility of carrot particles of different sizes (ranging from cell fragments up to large cell clusters), generated from raw as well as from gently and intensely cooked carrot samples, was determined. In the second part of the study, the effect of human mastication on the particle size reduction of raw as well as of gently and intensely cooked carrot samples was investigated in order to allow identification and validation of a technique that could mimic mastication during in vitro procedures. Results showed a strong dependency of the all-E-β-carotene bioaccessibility on the particle size for raw and gently cooked carrots. After intense cooking, on the other hand, a considerable amount of all-E-β-carotene could be released from cell fragments (smaller than a cell) as well as from small and large cell clusters. Hence, the importance of mechanical breakdown, and thus also of (in vitro) mastication, is dependent on the carrot sample that is considered (i.e., the extent to which the carrot sample has been thermally processed prior to the particle size reduction). Structural changes occurring during mechanical and thermal processing are hereby key factors determining the all-E-β-carotene bioaccessibility. The average particle size distribution curves of raw and cooked carrots, which were chewed by 15 persons, could be mimicked by mixing 50 g of carrots using a Grindomix (Retsch) at 2500 rpm during 5 s. Based on this scientific knowledge, the identified in vitro mastication technique was successfully integrated in the in vitro digestion procedure determining the all-E-β-carotene bioaccessibility of carrot samples.