The milk and mammary gland (MG) microbiome can be influenced by several factors, such as mode of delivery, breastfeeding, maternal lifestyle, health status, and diet. An increasing number of studies show a variety of positive effects of consumption of probiotics during pregnancy and breastfeeding on the mother and the newborn. The aim of this study was to investigate the effect of oral administration of probiotics Lactobacillus gasseri K7 (LK7) and Lactobacillus rhamnosus GG (LGG) during pregnancy and lactation on microbiota of the mouse mesenteric lymph nodes (MLN), MG, and milk. Pregnant FVB/N mice were fed skim milk or probiotics LGG or LK7 resuspended in skim milk during gestation and lactation. On d 3 and 8 postpartum, blood, feces, MLN, MG, and milk were analyzed for the presence of LGG or LK7. The effects of probiotics on MLN, MG, and milk microbiota was evaluated by real-time PCR and by 16S ribosomal DNA 454-pyrosequencing. In 5 of 8 fecal samples from the LGG group and in 5 of 8 fecal samples from the LK7 group, more than 1 × 10(3) of live LGG or LK7 bacterial cells were detected, respectively, whereas no viable LGG or LK7 cells were detected in the control group. Live lactic acid bacteria but no LGG or LK7 were detected in blood, MLN, and MG. Both probiotics significantly increased the total bacterial load as assessed by copies of 16S ribosomal DNA in MLN, and a similar trend was observed in MG. Metagenomic sequencing revealed that both probiotics increased the abundance of Firmicutes in MG, especially the abundance of lactic acid bacteria. The Lactobacillus genus appeared exclusively in MG from probiotic groups. Both probiotics influenced MLN microbiota by decreasing diversity (Chao1) and increasing the distribution of species (Shannon index). The LGG probiotic also affected the MG microbiota as it increased diversity and distribution of species and proportions of the genera Lactobacillus and Bifidobacterium. These results provide evidence that probiotics can modulate the bacterial composition of MLN and MG microbiota in ways that could improve the health of the MG and, ultimately, the health of the newborn.