Studies investigating carotene bioaccessibility (release from the food matrix to a solubilized form) directly from plant material during the process of digestion are scarce, mainly due to the difficulties associated with obtaining such material. Therefore, this paper examines the relationship between tissue microstructure and carotene bioaccessibility using an in vitro digestion model. Dietary oil provides a pool for the initial solubilization. Therefore, carotene partitioning into an emulsified oil phase was assessed using raw carrot tissue and carrot tissue subjected to various degrees of heating and particle size reduction and, in all cases, was found to be greatly reduced compared with juiced carrot. Carotene bioaccessibility was found to be greater from raw tissues than heated tissues of the same size. This is because heating increases the propensity for intact cells to separate, effectively encapsulating the carotene. Although the gross structure of the tissues was found to be relatively unaffected by in vitro digestion, at the cellular level some cell-wall swelling and cell death were observed, particularly close to the surfaces of the tissue. This study suggests that cell-wall rupture prior to digestion is an absolute requirement for carotene bioaccessibility in the upper intestine and that heating does not enhance carotene release from intact cells.