BackgroundThe combination of cultivation studies with molecular analysis approaches allows characterization of the complex human gut microbiota in depth. In vitro cultivation studies of infants living in rural sub-Saharan Africa are scarce. In this study, a batch cultivation protocol for Kenyan infant fecal microbiota was validated.MethodsFresh fecal samples were collected from 10 infants living in a rural area of Kenya. Samples were transported under protective conditions and subsequently prepared for inoculation within less than 30 h for batch cultivation. A diet-adapted cultivation medium was used that mimicked the daily intake of human milk and maize porridge in Kenyan infants during weaning. 16 S rRNA gene amplicon sequencing and HPLC analyses were performed to assess the composition and metabolic activity, respectively, of the fecal microbiota after 24 h of batch cultivation.ResultsHigh abundance ofBifidobacterium(53.4 ± 11.1%) and high proportions of acetate (56 ± 11% of total metabolites) and lactate (24 ± 22% of total metabolites) were detected in the Kenyan infant fecal microbiota. After cultivation started at an initial pH 7.6, the fraction of top bacterial genera (≥ 1% abundant) shared between fermentation and fecal samples was high at 97 ± 5%. However,Escherichia-Shigella,Clostridium sensu stricto1,BacteroidesandEnterococcuswere enriched concomitant with decreasedBifidobacteriumabundance. Decreasing the initial pH to 6.9 lead to higher abundance ofBifidobacteriumafter incubation and increased the compositional similarity of fermentation and fecal samples. Despite similar total metabolite production of all fecal microbiota after cultivation, inter-individual differences in metabolite profiles were apparent.ConclusionsProtected transport and batch cultivation in host and diet adapted conditions allowed regrowth of the top abundant genera and reproduction of the metabolic activity of fresh Kenyan infant fecal microbiota. The validated batch cultivation protocol can be used to study the composition and functional potential of Kenyan infant fecal microbiota in vitro.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-023-02915-9.