To explore the roles microbiome of urinary tract played in calcium oxalate stones (CaOx) formation, we collected two sides' pelvis urine of patients with unilateral CaOx stones to set self-control to diminish the influence of systemic factors. Patients with unilateral CaOx stones were recruited in our study according to strict criteria. 16S rRNA gene sequencing was applied to every pair of pelvis urine. Bacterial genome sequencing of Enterobacter cloacae was conducted and bioinformatic analysis was applied to explore the possible pathways of Enterobacter cloacae inducing CaOx stones formation. In vivo experiments were conducted to validate our claims. Von Kossa staining, TUNEL assay and Western Blot were applied to SD rats exploring the mechanism of stone formation. We found 26 significantly different bacteria between stone sides and non-stone sides' pelvis urine, among which Enterobacter cloacae ranked the most different. Bacterial genome sequencing of Enterobacter cloacae revealed that its virulence factors included Flagellin, LPS and Fimbrial. GO and KEGG analysis revealed it probably induced CaOx stone formation via ion binging and signaling transduction pathways. The results of animal experiments indicated that Glyoxylic Acid could promote apoptosis and crystal depositions of kidney comparing with control group while pre-injected with Enterobacter cloacae could apparently compound the effects. While Western Blot demonstrated that Glyoxylic Acid or Enterobacter cloacae could increase the expression of IL-6, Mcp-1, BMP2 and OPN in rats' kidney, Glyoxylic Acid and Enterobacter cloacae together could aggravate these increases. These findings indicated that Enterobacter cloacae might play important roles in CaOx stones formation. However, this study is just a preliminary exploration; further studies still need to be conducted.