The aim of this study was to identify the correlations of a common polymorphism (rs6774494 A > G) in the EVI-1 gene targeted by micro-RNA (miRNA)-206/133b with the pathogenesis of breast cancer (BC). A total of 196 unrelated ethnic Han Chinese women diagnosed with primary BC were consecutively recruited and 200 healthy controls were randomly selected from the same population-based cohort. Direct PCR sequencing assay was used to detection of rs6774494 A > G polymorphism in the EVI-1 gene. Real-time quantitative PCR (RT-PCR) analysis was performed to verify the alterations of the EVI1 messenger RNA (mRNA) levels. Kaplan-Meier analysis was used to investigate and to estimate the survival outcomes for each endpoint. All statistical analyses were performed with SPSS software (version 18.0, SPSS, Chicago, IL). Our results demonstrated that the carriers of EVI-1 AG genotype were more likely to develop BC when compared with the EVI-1 GG genotype (P = 0.034, OR = 1.26, 95% CI = 1.02 ∼ 1.57). In addition, it was found that patients with the G (AG + GG) allele of EVI-1 genetic variants were associated with higher risk of BC compared with the EVI-1 AA genotype (OR = 1.26, 95% CI = 1.02 ∼ 1.54, P = 0.028). The results of a subgroup analysis stratified by menopause revealed that in female post-menopause subgroup patients with the EVI-1 G allele were correlated with a higher risk of BC than those with the EVI-1 AA genotype (OR = 1.31, 95% CI = 1.00 ∼ 1.72, P = 0.054). Kaplan-Meier analyses suggested that carriers of the G allele (AG + GG) were associated with poorer overall survival (OS) and progression-free survival (PFS) compared with those with AA genotype (OS P = 0.042; PFS P = 0.036, respectively). The correlation analysis showed that EVI-1 mRNA levels were negatively associated with miRNA-206/133b levels in the carriers of the G allele (AG + GG) (r = -1.274, P < 0.05). Our findings provide evidence that the EVI-1 rs6774494 G > A polymorphism targeted by miRNA-206/133b may contribute to the pathogenesis of BC.