Recent advances in novel culturing methods and high-resolution sequencing have provided significant insight into the composition and function of the human gut microbiota. Despite the known associations between the microbiota and the host immune response, there is an unmet need to define these interactions, particularly in disorders with a complex pathophysiology such as inflammatory bowel disease (IBD). Site-specific mucosal culturing of intestinal biopsies has enabled the establishment of a human gut microbiome culture collection from patient cohorts. Host gene transcriptional analysis and bacterial metagenomic sequencing identified key gut microbiota subclades that are associated with an inflammatory gene signature. To experimentally validate these findings in vitro, mucosal bacterial isolates were co-cultured with intestinal epithelial cells which resulted in a cell cytotoxic phenotype and transcription of host genes belonging to the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathways. Epithelial cell cytotoxicity was subsequently observed following bacterial co-culture in Transwell assays and similarly in response to conditioned media from bacterial-stimulated epithelial cells. This indicates that there may be specific microbial factors or metabolites that function as mediators of innate immune signalling and highlights the importance of microbiota-host cell crosstalk. Further studies will provide a better understanding of microbiota-host interactions in IBD and other microbiota-associated disorders, unlimited to the gastrointestinal tract.