Transplantation of germ-free (GF) mice with microbiota from mice or humans stimulates the intestinal immune system in disparate ways. We transplanted a human microbiota into GF C57BL/6 mice and a murine C57BL/6 microbiota into GF C57BL/6 mice and Swiss-Webster (SW) mice. Mice were bred to produce an offspring generation. 56% of the Operational Taxonomic Units (OTUs) present in the human donor microbiota established in the recipient mice, whereas 81% of the C57BL/6 OTUs established in the recipient C57BL/6 and SW mice. Anti-inflammatory bacteria such as Faecalibacterium and Bifidobacterium from humans were not transferred to mice. Expression of immune-related intestinal genes was lower in human microbiota-mice and not different between parent and offspring generation. Expression of intestinal barrier-related genes was slightly higher in human microbiota-mice. Cytokines and chemokines measured in plasma were differentially present in human and mouse microbiota-mice. Minor differences in microbiota and gene expression were found between transplanted mice of different genetics. It is concluded that important immune-regulating bacteria are lost when transplanting microbiota from humans to C57BL/6 mice, and that the established human microbiota is a weak stimulator of the murine immune system. The results are important for study design considerations in microbiota transplantation studies involving immunological parameters. The gut microbiota is an important component of human health. For studying its role in health and disease, aiming for the development of microbiota-targeting therapeutics, food products and ingredients, mice transplanted with human microbiotas (HMs) have been described and applied for several decades 1-6 , although concerns pertaining to limitations of this model system have been raised 7-10. Often such models are only studied on their phenotypic expression and the microbiota is not characterized, or the opposite is the case 8. Laboratory rodents are routinely raised in specific pathogen free (SPF) barrier facilities strictly protected from their wild conspecifics. For many years the microbial starting point for many rodent breeding colonies has been the Altered Schaedler Flora (ASF) or variants thereof. ASF consists of eight specific bacterial strains originating from conventional laboratory mice from the 1960's and 1970's, and it is therefore considered mouse-specific 11,12. In addition to this, laboratory rodents are exposed to microbes deriving from human staff in the facility, at least if bedding, food, and other materials are sterilized before introduced to the facility. Evolutionary adaptation to the host environment may drive formation of mouse-specific species and strains originally derived from humans, as