The human multidrug resistance gene 1 ( MDR1 ) encodes a plasma membrane, P-glycoprotein (Pgp), which functions as the transmembrane efflux pump for various structurally unrelated anticancer agents and toxins. Polymorphisms in the MDR1 gene may have an impact on the expression and function of Pgp, thereby influencing the susceptibility to various diseases, including cancer. Recently, a silent C3435T polymorphism in exon 26 of MDR1 has been reported to be associated with decreased expression of Pgp in TT genotype carriers and thus it may alter the physiological protective role of Pgp and influence disease risk. To evaluate the association of this polymorphism with breast cancer, 106 patients with breast cancer and 77 healthy controls were enrolled in this study. They were visited at two centers during a 1-year period (2006–2007). Data about the risk factors of breast cancer were collected using questionnaires. DNA of the whole-blood sample was extracted, and the polymorphic fragment was amplified by polymerase chain reaction using specific primers. The C3435T polymorphism was detected by the restriction fragment length polymorphism method. There were no significant differences in genotype ( p = 0.744) and allele ( p = 0.590) frequencies between patients and control subjects. Moreover, distribution of the breast cancer patients' risk factors was not different among CC, CT, and TT genotypes. Our results suggest that C3435T MDR1 polymorphism was not associated with the susceptibility to breast cancer in the population studied.