Infertility is defined as the inability of the couples to achieve pregnancy after 12 months of unprotected intercourse. P-glycoprotein (P-gp) also known as multidrug resistance protein 1 (MDR1) is an important protein of the cell membrane that pumps many foreign substances out of cells and has a protective role in sensitive tissues such as testis. MDR1 gene is located on q21.1 of chromosome 7. Hence, this study aimed to assess the association of MDR1 polymorphism and idiopathic male infertility. In this study (case- control), DNA was extracted from blood leukocytes of 136 male patients with idiopathic infertility as well as 130 healthy men. Genotypes were determined by using PCR-RFLP technique and EcoO109I enzyme. The genotypes frequencies of CC, CT and TT in patient group were 19.12%, 39.70% and 41.18%, respectively, and the genotype frequencies of CC, CT and TT in control group were 12.30%, 61.54% and 26.16%, respectively. The study findings revealed that a significant association was found between MDR1 polymorphism and idiopathic infertility (P= 0.001). Therefore, the results suggest that CT heterozygous genotype has a protective effect on male fertility (P= 0.01, OR= 0.41; 95%CI: 0.23- 0.84). However, to achieve more accurate results, it is necessary to examine a larger target population.