Display omitted •Supplementation of L-NCFM and B-LBi07 regulate host metabolism and gut microbiota metabolism.•Supplementation of L-NCFM and B-LBi07 increase beneficial bacteria and inhibit harmful bacteria.•Supplementation of L-NCFM and B-LBi07 promote the functional maturation of gut bacterial community.•Key Metabolites are strongly correlated with the gut microbiota composition. To investigate the effect of Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 (1:1) on microbiota composition and function, metabolism as well as microbiota-metabolite relationships of weaned rats, four groups of male Sprague-Dawley rats were orally supplemented with either probiotics (2.5 × 1010 cfu/kg, 5.0 × 1010 cfu/kg, 1.0 × 1011 cfu/kg) or vehicle (water) for 28 days. Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 treatment increased levels of glutamate, short chain fatty acids and ketone bodies, and decreased levels of choline, creatine and lactate. Levels of Rothia, Bifidobacterium, Jeotgalicoccus, Lactobacillus, Acinetobacter and Oligella were significantly increased, whereas Parabacteroides, Enterococcus and Candidatus Arthromitus were reduced. The PICRUSt analysis indicated Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 could promote the functional maturation of gut bacterial community. Strong correlations were observed between metabolites and Parabacteroides, Enterococcus, Candidatus Arthomitus, Bifidobacterium, Jeotgalicoccus, Lactobacillus and Acinetobacter. Collectively, supplementation of Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 helps promote the structure and functional maturation of intestinal microbiome in weaned rats.