Studies on the impact of colonic fermentation of plant sterol (PS)-enriched foods using dynamic in vitro models are limited. This study aims to evaluate the effect of a 72h-dynamic in vitro digestion-colonic fermentation (using the simgi® system) of a PS-enriched rye bread on colonic microbial population, and short chain fatty acids (SCFA) and ammonium production. In all colon compartments (ascending colon (AC), transverse colon (TC), and descending colon (DC)) (72 vs. 0h), a reduction in ammonium concentration (5.7-9.4-fold) and an increase in Staphylococcus spp. (1.4-2.1-fold), Lactobacillus spp. (1.5-fold), Bifidobacterium spp. (1.3-1.5-fold), and Enterococcus spp. (1.4-1.6-fold) was observed. In AC and TC, total SCFA decreased (4- and 1.3-fold, respectively), while increased in DC 1.4-fold due to an increment of butyrate content from 4 to 45mM. These results suggest that PS-enriched rye bread favors the growth of beneficial microbial species and production of butyrate, a fuel source for the enterocyte, promoting health benefits.