Osteopontin (OPN) is one of the urinary proteins with an important role in stone formation. Recently, OPN Ala250 (rs1126616) polymorphism and other single nucleotide polymorphisms (SNPs) have been studied to define their role in urolithiasis. This study was conducted to examine the impact of OPN Ala250 polymorphism on the risk of stone formation and their association with serum OPN levels. OPN Ala250 polymorphism was investigated in 127 urolithiasis patients and 92 healthy controls. Stones were analyzed for their chemical composition by using X-Ray diffraction method. Genomic DNA was isolated from peripheral blood leucocytes. The study groups were genotyped by PCR-RFLP and serum OPN levels were measured by ELISA. There was a significant difference between urolithiasis patients and controls concerning genotype and allele frequencies of OPN Ala250 (p<0.05). Separate analysis by BMI greater or less than 25 kg/m(2) showed that the presence of one mutant T-allele was more frequent in patients with higher BMI than patients with BMI less than 25 kg/m(2) (p<0.05). Serum OPN concentrations were two-fold higher in the control group compared to urolithiasis patients (p<0.05). But the mean serum levels did not show any significant difference between OPN Ala250 genotypes in both groups. Moreover, we found an association between higher BMI and stone formation. Our findings suggest that OPN Ala250 polymorphism is associated with the correlation between weight gain and urolithiasis. However, the correlation between urolithiasis and obesity needs to be further studied in larger cohorts.