AbstractGut microbiota development in formula‐fed and breast‐fed infants is known to differ. This could relate to the usage of unmodified vegetable oil instead of mammalian fat in infant formula (IF), causing the enhanced formation of the poorly soluble soap calcium palmitate (CP) in the infant's gut. Here we investigatein vitrothe possible influence of CP on the infant gut bacteria. The growth of several bacterial species dominant in the infant's gut was analyzed by culturing in media with CP.Faecalibacterium prausnitziias a sensitive representative was analyzed in detail by scanning transmission electron microscopy, membrane staining, gas chromatography, and microbial fuel cell experiments. Of all bacteria tested, the growth of several bifidobacteria andF. prausnitziiwas reduced at 0.01 mg/ml CP,Bifidobacterium infantisstopped growing completely. CP reduced the cell envelope thickness ofF. prausnitzii, disturbed the cell membrane fatty acids and function of membrane proteins involved in electron transport. CP inhibited the growth of bifidobacteria and faecalibacteria. This suggests that modification of fat in IF may benefit the development of the gut microbiota in formula‐fed infants by supporting the colonization of important beneficial bacteria in early life. Future clinical studies are needed to confirm this.