The Twin Simulator of the Human Intestinal Microbial Ecosystem (TWINSHIME®) was initially developed to study the luminal gut microbiota of the ascending (AC), transverse (TC), and descending (DC) colon regions. Given the unique composition and potential importance of the mucosal microbiota for human health, the TWINSHIME was recently adapted to simulate the mucosal microbiota as well as the luminal community. It has been previously demonstrated that the luminal community in the TWINSHIME reaches a steady state within two weeks post inoculation, and is able to differentiate into region specific communities. However, less is known regarding the mucosal community structure and dynamics. During the current study, the luminal and mucosal communities in each region of the TWINSHIME were evaluated over the course of six weeks. Based on 16S rRNA gene sequencing and short chain fatty acid analysis, it was determined that both the luminal and mucosal communities reached stability 10-20 days after inoculation, and remained stable until the end of the experiment. Bioinformatics analysis revealed the formation of unique community structures between the mucosal and luminal phases in all three colon regions, yet these communities were similar to the inoculum. Specific colonizers of the mucus mainly belonged to the Firmicutes phylum and included Lachnospiraceae (AC/TC/DC), Ruminococcaceae and Eubacteriaceae (AC), Lactobacillaceae (AC/TC), Clostridiaceae and Erysipelotrichaceae (TC/DC). In contrast, Bacteroidaceae were enriched in the gut lumen of all three colon regions. The unique profile of short chain fatty acid (SCFA) production further demonstrated system stability, but also proved to be an area of marked differences between the in vitro system and in vivo reports. Results of this study demonstrate that it is possible to replicate the community structure and composition of the gut microbiota in vitro. Through implementation of this system, the human gut microbiota can be studied in a dynamic and continuous fashion.