Purpose: To establish a male Wistar rat model of nanobacteria (NB)-induced kidney stones. Methods: Sixty male Wistar rats were randomly divided into control group (NC group) given caudal vein injection of saline + saline gavage, and NB-induced stone group (NBS group) given caudal vein injection of NB + saline gavage. Results: Compared with NC, serum creatinine, blood uric acid, urea nitrogen and urinary calcium levels in NBS group increased between weeks 3 and 8 (p < 0.05). Kidney index (kidney weight/body weight ratio) in the NBS group was higher than that in NC group from weeks 8-10. At week 8, urine pH and serum phosphorus in NBS group were higher than those in NC group (p < 0.05). Between weeks 6 and 7, serum calcium in NBS group was higher than that in NC group (p < 0.05). Calcium crystals in NBS rats were distributed mostly in the distal and proximal convoluted tubules. However, no such crystals were observed in NC rats. Similarly, no such pathological changes were seen in the renal tissue of NC group. Calculus analysis showed that stone formation was higher in NBS group than in NC group (p < 0.05). There was no significant difference in micro-CT between the two groups (p > 0.05). Conclusion: The successful establishment of the Wistar rat kidney stone model using NB cultured from urine of upper urinary tract stone patient is potentially useful for further etiological studies on kidney stone formation.