Objective To address the effects of osteopontin (OPN) on calcium oxalate renal calculus formation. Methods We silenced the OPN gene in rat kidney in vivo using lentivirus vector-deliverd short hairpin RNA (shRNA) targeting OPN through renal venous transduction. On the day 7 after injection with lentivirus, rats were fed on water containing 0.8% glycol and 1% ammonium chloride for 9 days. At the end of the experiments, the rats were killed and the kidney tissues were removed for subsequent analysis. Results The lentivirus vector could effectively infect renal cortex and medulla tissue. OPN-shRNA transfection resulted in significant reduction in the content of OPN. Crystals recorded as the average total pixels (object area) were 20 716±7 755 and 54 921±3 469 in renal cortex sections in the OPN-shRNA group and negative lentivirus vector group, respectively (P<0.01), as well as 18 618±2 285 and 30 475±1 735 in the medulla sections in both groups, respectively (P<0.01). Conclusion Specific knockdown of OPN gene in kidneys of hyperoxaluric rats leads to a decrease in OPN production and significantly inhabits calcium oxalate crystal deposition. OPN may play a certain role in promoting calcium oxalate renal calculus formation. Key words: Osteopontin; Calcium oxalate; Nephrolithiasis; RNA interference