An important process in kidney stone formation is the conversion of retentive crystals in renal tubules to concrete stones. Osteopontin (OPN) is the major component of the kidney calcium-containing stone matrix. In this study, we estimated OPN function in early morphological changes of calcium oxalate crystals using OPN knockout mice: 100 mg/kg glyoxylate was intra-abdominally injected into wildtype mice (WT) and OPN knockout mice (KO) for a week, and 24-h urine oxalate excretion showed no significant difference between WT and KO. Kidney crystal depositions were clearly detected by Pizzolato staining but not by von Kossa staining in both genotypes, and the number of crystals in KO was significantly fewer than in WT. Morphological observation by polarized light optical microphotography and scanning electron microphotography (SEM) showed large flower-shaped crystals growing in renal tubules in WT and small and uniform crystals in KO. X-ray diffraction detected the crystal components as calcium oxalate monohydrate in both genotypes. Immunohistochemical staining of OPN showed that the WT crystals contained OPN protein but not KO crystals. We concluded that OPN plays a crucial role in the morphological conversion of calcium oxalate crystals to stones in mouse kidneys.