Little is known regarding the interplay between microbiota and pancreas functions in humans as investigations are usually limited to distal sites, namely the analyses of fecal samples. The aim of this study was to investigate both ileal and fecal microbiota in response to pancreatic enzyme replacement therapy (PERT) in a porcine model of exocrine pancreatic insufficiency (EPI). PERT was stopped for ten days in ileo-cecal fistulated minipigs with experimentally induced EPI (n = 8) and ileal digesta as well as fecal samples were obtained before withdrawal, during withdrawal and after the reintroduction of PERT. Profound community changes occurred three days after enzyme omission and were maintained throughout the withdrawal phase. A reduction in α-diversity together with relative abundance changes in several taxa, in particular increases in Bifidobacteria (at both sites) and Lactobacilli (only feces) were observed. Overall, dysbiosis events from the ileum had accumulating effects in distal parts of the gastrointestinal tract with additional alterations occurring only in the colon. Changes were reversible after continuing PERT, and one week later, bacterial communities resembled those at baseline. Our study demonstrates the rapid and profound impacts of enzyme withdrawal in bacterial communities, contributing to our understanding of the interplay between pancreas function and microbiota.