Some of the single-nucleotide polymorphisms in miRNA genes have been studied to date to find their association with the risk of breast cancer (BC). However, no study has been conducted to investigate the association of the mir-520f rs75598818G>A in BC. In the present study, rs75598818 association with BC in an Iranian population has been investigated, and an in silico analysis was performed to predict the function of rs75598818 polymorphism in BC. The rs75598818 was genotyped in 129 BC patients and 144 healthy women, using the PCR-RFLP method. The frequency of alleles and genotypes were considered to find the associations between rs75598818 alleles/genotypes, and BC risk and pathological characteristics of the patients. Statistical analysis showed that the rs75598818 GA genotype was significantly associated with BC (GA versus GG, OR=0.50, 95% CI: 0.25–0.98, P =0.041), highstage BC (stage III/IV versus I/II, GA versus GG, OR=0.27, 95% CI: 0.09–0.81, P =0.015), and HER-2 positive status (GA versus GG, OR=19.00, 95% CI: 4.64–77.82, P <0.001). Notably, the rs75598818 GA genotype has a negative association pattern since it reduces the risk of BC and high stage BC. Conversely, it increases the risk of HER-2 positivity. Computational results suggested that the rs75598818 polymorphism affects the stability of mir-520f stem-loop and as a result miR-520f-3p production that is a potential tumour suppressor. A contribution of the mir-520f rs75598818 polymorphism to BC had been unexplored before. In the present study, we performed an association study and a bioinformatics approach to evaluate this polymorphism in BC. However, further functional experiments and large-scale association studies with various ethnicities are required to elaborate our findings.