The aim of this study was to investigate the effect of a functional probiotic cheese (FPC) on gut microbiota (GM), after simulated digestion performed by a multi-unit in vitro colon model (MICODE). Squacquerone-like cheese was produced using the starter Streptococcus thermophilus (control, CTRL), and supplemented with the probiotic Lacticaseibacillus rhamnosus, which was either subjected to high pressure homogenization (LrH) or not (Lr). Samples were stratified by cheese type, storage time, and colonic fermentation phase. Samples were then digested with MICODE and digests were characterized for ecological and functional profiles. The lactobacilli detected in Lr and LrH cheeses (9.0 log CFU/g) were represented by the probiotic strain L. rhamnosus and remained unchanged after storage at 4 °C. Lactobacilli levels in CTRLs increased from 1.5 log CFU/g to 2.0 log CFU/g after six days at 4 °C, while total coliforms remained below 1.5 log CFU/g in all samples. Real-time qPCR indicated a positive GM response after FPC simulated digestion, highlighting an abundance of bifidobacteria, lactobacilli and Clostridium group IV in LrH samples. Metataxonomy revealed higher levels of Firmicutes and Proteobacteria (p ≤ 0.05) after simulated digestion, as well as Megasphaera, Escherichia, Prevotella and Dorea. Moreover, an increase of short and medium chain fatty acids were detected by metabolomics. Overexpression of inferred KEGG metabolic pathways showed mainly fatty acids, novobiocin and amino acid metabolism. Understanding how functional foods can modify the GM may lead to the development of targeted microbiome-based therapies and the exploitation of these foods for the benefit of human health.