Weaned piglets are vulnerable to nutritional, physiological, and psychological stressors, leading to abrupt taxonomic and functional shifts in the intestinal microbiome. In this study, an integrated approach combination of 16S rDNA gene sequencing and the mass spectrometry-based metabolomics techniques was used to investigate the effects of weaning stress on intestinal microbial composition and its metabolic profiles of piglets. Three litters of suckling piglets with same parity were chosen. The samples of colonic contents were collected from each selected piglets (weaned day, 3 days after weaned) for microbial and metabolomics analysis. The results showed that Lachnospiraceae, Negativicutes, Selenomonadales, Campylobacterales and other 15 species increased after weaning, while Porphyromonadaceace, Alloprevotella, Barnesiella and Oscillibacter decreased. Based on the function profiles prediction and metabolomic analysis, five key metabolic pathways including Phenylalanine metabolism, Citrate cycle (TCA cycle), Glycolysis or Gluconeogenesis, Propanoate metabolism, Nicotinate and nicotinamide metabolism might be the relevant pathways involved in weaning stress-induced gut microbiota dysbiosis. Taken together, these results indicated that weaning stress not only changed microbial composition and function but altered the microbial metabolic profiles in the intestine, which might provide a new insight in alleviating weaning stress and facilitating disease prevention during the period of weaning in piglets.