Gut microbiota play a critical role in infant health. It is now accepted that breastmilk contains live bacteria from endogenous and exogenous sources, but it remains unclear whether these bacteria transfer to the infant gut and whether this process is influenced by breastmilk feeding practices. Here, we show that certain bacteria, including Streptococcus spp. and Veillonella dispar, co-occur in mothers’ milk and their infants’ stool, and co-occurrence is reduced when infants receive pumped breastmilk. The relative abundances of commonly shared species are positively correlated between breastmilk and stool. Overall, gut microbiota composition is strongly associated with breastfeeding exclusivity and duration but not breastmilk feeding mode (nursing versus pumping). Moreover, breastmilk bacteria contributed to overall gut microbiota variation to a similar extent as other modifiers of the infant microbiome, such as birth mode. These results provide evidence that breastmilk may transfer bacteria to the infant gut and influence microbiota development. Display omitted •Breastfeeding exclusivity and duration strongly influence infant gut microbiota•A few bacterial species co-occur in mothers’ milk and their infants’ stool•This co-occurrence is reduced when breastmilk is pumped and fed from a bottle•Bacteria in breastmilk contribute to overall infant gut microbiota composition Fehr et al. analyze infant gut microbiota in the CHILD cohort and identify associations with breastmilk feeding practices (exclusivity, duration, and pumping) and breastmilk microbiota. Within mother-infant dyads, co-occurrence of bacteria in breastmilk and stool suggests bacteria in breastmilk may transfer to the infant and influence the developing gut microbiota.