The impact of an early food introduction on the microbiota composition and microbial metabolism in colon was investigated using a new-born piglet model. At day 4 after birth, 10 litters of piglets were randomly allocated to a sow-rearing group (SR group) and a milk-replacer supplementing group (MRS group) (n = 5). A commercial milk replacer was given to the suckling piglets in the MRS group from the 4th day to the 28th day. Pyrosequencing of the V3-V4 region of the 16S rRNA genes showed that the milk replacer supplementation significantly decreased the relative abundance of Lactobacillus, Clostridium XI, Blautia, Clostridium sensustricto and Escherichia (p = 0.08) in the colon of the piglets, but significantly increased the relative abundance of Paraprevotella on the 28th day. In addition, the abundance of Rumminococcus, Clostridium XlVa, Succiniclasticum, Clostridium IV tended to increase in the MRS group. The concentrations of acetate, propionate, butyrate, valerate and branch-chain fatty acids (BCFAs) in the colonic digesta increased with the milk replacer supplementary in the MRS group. In addition, the milk replacer supplementary increased the expression level of Toll-like receptor 4 (TLR4), but decreased the expression level of interleukin-6 (IL-6) in the colonic mucosa of the piglets. In conclusion, an early food introduction can influence the gut bacterial composition and metabolism, and may further affect the intestinal health by modifying the gene transcription related to the colonic function. These findings may provide some guidelines for the early nutrition supplementation for infants during the lactation period.