The prevention of cardiovascular disease using foods fortified with plant sterols (PS), with a hypocholesterolemic effect, is important for the elderly population. This study aimed at identifying the different PS present in PS-enriched wholemeal rye bread (WRB) and in the ingredient source of PS, to evaluate their bioaccessibility in WRB by simulated static digestion. The gastrointestinal conditions of the elderly were adapted, and the results were compared with the adult population. Nine PS were identified, and a total amount of 2.18 g/100 g WRB was determined. Bioaccessibility was reduced in the elderly model with gastrointestinal adaptation vs. the adult model (11.2 vs. 20.3%), but no differences were observed when adapting only the gastric phase. Even though there was lower bioaccessibility of PS in the elderly, they could benefit from the consumption of WRB as it has a good nutritional profile. Further investigation including in vivo assays is needed to strengthen the results.