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Changes in the human gastrointestinal microbiome are associated with several diseases. To infer causality, experiments in representative models are essential, but widely used animal models exhibit limitations. Here we present a modular, microfluidics-based model (HuMiX, human–microbial crosstalk), which allows co-culture of human and microbial cells under conditions representative of the gastrointestinal human–microbe interface. We demonstrate the ability of HuMiX to recapitulate  in vivo  transcriptional, metabolic and immunological responses in human intestinal epithelial cells following their co-culture with the commensal  Lactobacillus rhamnosus  GG (LGG) grown under anaerobic conditions. In addition, we show that the co-culture of human epithelial cells with the obligate anaerobe  Bacteroides caccae  and LGG results in a transcriptional response, which is distinct from that of a co-culture solely comprising LGG. HuMiX facilitates investigations of host–microbe molecular interactions and provides insights into a range of fundamental research questions linking the gastrointestinal microbiome to human health and disease.

A microfluidics-based in vitro model of the gastrointestinal human–microbe interface