Objectives: We aimed to investigate the effect of antibiotics-induced microbiota depletion on AKI-to-CKD transition after ischemia-reperfusion injury. Methods: We established AKI-to-CKD transition mice model after unilateral IRI (27 minutes) and maintained for 4 weeks. For AIMD, C57BL/6 mice was treated with a mixture of 4 broad-spectrum antibiotics (neomycin 25 mg/kg, metronidazole 25 mg/kg, ampicillin 60 mg/kg, and vancomycin 400 mg/kg) via oral gavage for 2 weeks before and after unilateral IRI.   Results: Mice with AIMD after unilateral IRI showed significantly decreased extent of kidney tissue fibrosis assessed by Masson’s trichrome (18.7% ± 9.8 % vs. 4.7% ± 2.9 %, P = 0.02) and Sirius red (20.1% ± 8.1% vs. 5.9% ± 3.3%, P = 0.005) staining. Immunohistochemical staining for Nox2 showed decreased extent of Nox2 in tubular area after AIMD compared to the controls (20.4% ± 4.2% vs. 5.0% ± 2.1%, P <0.001). Areas of CD3 positive T cell areas decreased significantly after AIMD (0.67% ± 0.54% vs. 0.24% ± 0.16%, P = 0.04). In RT-PCR, TGFβ (4.2 ± 3.3 fold vs. 1.4 ± 1.0 fold, P = 0.03), periostin (2.6 ± 1.6 fold vs. 0.9 ± 0.4 fold, P = 0.003), and IL-18 expression (2.7 ± 1.7 fold vs. 0.9 ± 0.4 fold, P = 0.003) decreased after AIMD. In metabolomics profile and microbiota analysis, AIMD inhibited unilateral IRI induced increase of propionate, betaine, methylamine, N,N-dimethylglycine, trimethylamine, and trimethylamine N-oxide (TMAO), and AIMD induced alteration of metabolomic profiles were associated with decreased actinobacteria, gemmatimonadetes, cyanobacteria, and armatimonadetes. Conclusions: AIMD successfully altered intestinal microbiota and metabolomic profiles associated with kidney disease outcomes. AIMD-induced alteration in intestinal microbiota and metabolomic profiles attenuates AKI-to-CKD transition and kidney fibrosis via inhibition of inflammatory response and reactive oxygen species.