In this paper, the mouse pup-in-a-cup model was improved for younger mouse pups, and the effect of artificial rearing on gut microbiota development was evaluated. Intragastric cannulas were placed through the esophagus into 3-day-old C57BL/6J mice (n=48), and the mice were artificially reared (AR) with mouse milk substitute (MMS). Littermate pups (n=20) were maternally reared (MR) as controls. The feces of 3-day-old pups were analyzed by combining the PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting technique and sequencing of 16s rRNA gene fragments. After 11 days of artificial rearing, 37 of 48 pups were still alive. There were no significant changes in the number of DGGE bands or the Shannon index between the two groups. However, several bands in the AR group were obviously different from those in the MR group in the DGGE profile. These results demonstrate that it is possible to implant intragastric cannulas into 3-day-old C57BL/6J mice pups. However, the variation in the gut microbiota composition is non-negligible, even though the AR pups grow well.