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Background Solute-linked carrier 26 gene family 6 (SLC26A6), which is mainly expressed in intestines and kidneys, is a multifunctional anion transporter crucial in the transport of oxalate anions. This study aimed to investigate the role of kidney SLC26A6 in urolithiasis. Methods Patients were divided into two groups: stone formers and nonstone formers. Samples were collected from patients following nephrectomy. Lentivirus with Slc26a6 (lentivirus-Slc26a6) sequence and lentivirus with siRNA-Slc26a6 (lentivirus-siRNA-Slc26a6) sequence were transfected into rats’ kidneys respectively and Slc26a6 expression was detected using Western blot and immunohistochemical analyses. After administering ethylene glycol, oxalate concentration and prevalence of stone formation between the transgenic and control groups were measured using 24-h urine analysis and Von Kossa staining, respectively. Results Immunohistochemical and Western blot analyses indicated that stone formers had a significantly higher level of expression of SLC26A6 in the kidney compared with the control group. After lentivirus infection, the urinary oxalate concentration and rate of stone formation in lentivirus-Slc26a6-tranfected rats increased remarkably, while lentivirus-siRNA-Slc26a6-transfected rats showed few crystals. Conclusion The results showed that high expression levels of renal SLC26A6 may account for kidney stone formation. Downregulating the expression of SLC26A6 in the kidney may be a potential therapeutic target to prevent or treat urolithiasis.