In this study, we investigated the impact of neonatal amoxicillin treatment on the development of the murine intestinal Lactobacillus community. Suckling BALB/c mice received a daily intragastric gavage of amoxicillin or saline from postnatal day 7 (PND 7) to PND 20. Just after the treatment (PND 21) and 5 weeks later (PND 56), the colon digesta samples were analyzed by Lactobacillus-specific quantitative real-time PCR analysis and PCR-denaturing gradient gel electrophoresis (DGGE) technique. Real-time PCR results showed that the levels of lactobacilli in the treatment group were similar to those in the control at PND 56. However, in DGGE analysis the number of DGGE bands and Shannon index were decreased significantly in comparison with control (P < 0.05). The dominant Lactobacillus strain in the murine colon changed from L. johnsonii to L. murinus. These results demonstrated that neonatal amoxicillin treatment led to a significant impact on the biodiversity of the murine intestinal Lactobacillus community within a long-time period.