A dynamic gastrointestinal digestion system (simgi) after a human oral phase was used, for the first time, to assess the bioaccessibility of plant sterols (PS) from wholemeal rye bread (74.8 ± 2.2 mg of PS/100 g d.m.) and PS-enriched wholemeal rye bread (PS-WRB) (1.6 ± 0.04 g of PS/100 g of fresh bread). The use of these solid food matrices requires a novel adaptation of the gastric phase of the system. The PS identified in the breads are campesterol, campestanol, stigmasterol, β-sitosterol, sitostanol, Δ5-avenasterol, Δ5,24-stigmastadienol, Δ7-stigmastenol, and Δ7-avenasterol. The bioaccessibility of the total PS, only quantifiable in PS-WRB, is 19.9%, with Δ7-avenasterol being the most bioaccessible and Δ5-avenasterol being the least (p < 0.05). As shown in this study, PS-WRB can be considered to be a good choice to include in the daily diet. Furthermore, although the use of dynamic digestion methods for evaluating bioaccessibility implies high costs and technical complexity, their application means a closer approximation to in vivo scenarios.