Human milk fat contains high concentrations of medium-chained fatty acids (MCFA) and triacylglycerols emulsified by a sphingomyelin-rich phospholipid membrane (milk phospholipids, MPL). Infant formula comprises mainly long-chained fatty acids (LCFA) emulsified with dairy proteins and soy lecithin (SL) lacking sphingomyelin. Sphingomyelin content and saturation level of phospholipids affect the gut lipase activity, which alters the concentrations of lipid hydrolysis products in ileum and colon, and hereby putatively affects the competitive advantage of specific gut bacteria. Thus, differences in phospholipid and FA composition may modulate the establishment of the gut microbiota. We investigated effects of fatty acid (FA) composition and emulsification (MPL vs SL) ingested during establishment of human gut microbiota in germ-free mice, and found that cecal microbiotas from mice given MCFA-rich emulsions were characterized by high relative abundances of Bacteroidaceae and Desulfovibrionaceae, while LCFA-rich emulsions caused higher abundances of Enterobacteriaceae, Erysipelotrichaceae, Coriobacteriaceae and Enterococcaceae. Consumption of SL-emulsified lipids skewed the community towards more Enterococcaceae and Enterobacteriaceae, while MPL increased Bacteroidaceae, Desulfovibrionaceae, Rikkenellaceae and Porphyromonadaceae. Intake of SL increased cecal concentrations of iso-valeric and iso-butyric acids. This suggests that fat-type and emulsifiers applied in infant formula may have distinct effects on the establishment of the gut microbiota in formula-fed infants.