Abstract: Objective To explore the relationship between the duration of antibiotic use after birth and gut microbiota dysbiosis in extremely preterm infants. Methods A total of 29 preterm infants with gestational ages under 32 weeks who were hospitalized at the First Affiliated Hospital of Nanjing Medical University from February 2023 to September 2023 were prospectively included. According to the duration of antibiotic use, the infants were divided into two groups: a ≤7-day group (antibiotics used for 6-7 days, n=12) and a >7-day group (antibiotics used for 8-28 days, n=17). Fecal samples were collected at 7, 14, 21, and 28 days after birth. Gut microbiota was analyzed by 16S rDNA high-throughput sequencing. Their clinical data, gut microbiota, and short-chain fatty acids (SCFAs) were analyzed by bioinformatics, Wilcoxon rank-sum tests, t-tests, or Fisher's exact tests. Results Both groups shared the same clinical features. The duration of antibiotic use within 28 days was 7.0 (6.0, 7.0) days for the ≤7-day group and 13.0 (9.8, 19.3) days for the >7-day group (Z=-4.61, P<0.001). A total of 112 fecal samples were analyzed for 16S rDNA and 20 samples for SCFAs. On day 7, no statistical differences were found between the two groups at the phylum, class, order, family, genus, or species levels, nor in the Chao, ACE, or Shannon diversity indices. On day 14, significant differences emerged: the relative abundance of Verrucomicrobiota (P=0.030) and Clostridium sensu stricto (P=0.039), as well as acetate concentration (P=0.015), decreased in the >7-day group. On day 21, differences persisted, and the relative abundance of Verrucomicrobiota remained decreased (P=0.020), while the relative abundance of Actinobacteria (P=0.048) and Shannon diversity index (P=0.018) increased in the >7-day group. On day 28, these differences disappeared, and diversity showed no statistical significance between the groups. Within-group comparisons in the ≤7-day group revealed a significant decline in the relative abundance of Bacteroidetes (P=0.026), Streptococcus thermophilus (P=0.023), and Burkholderia (P=0.026) from 7 to 14 days after birth, without statistical changes in diversity. From 14 to 28 days after birth, no statistical differences were observed at any taxonomic level. For the >7-day group, no statistical within-group differences were found from 7 to 14 days or from 14 to 28 days. Conclusions The gut microbiota of the two groups is similar on day 7. Due to the difference in antibiotic durations, statistical differences in gut microbiota were observed on 14 and 21 days after birth. But by 28 days after birth, the gut microbiota of both groups had "recovered" to similar levels.