Abstract Introduction Single-nucleotide polymorphisms (SNPs) at 6q25.1 that are associated with breast cancer susceptibility have been identified in several genome-wide association studies (GWASs). However, the exact causal variants in this region have not been clarified. Methods In the present study, we genotyped six potentially functional single-nucleotide polymorphisms (SNPs) within the CCDC170 and ESR1 gene regions at 6q25.1 and accessed their associations with risk of breast cancer in a study of 1,064 cases and 1,073 cancer-free controls in Chinese women. The biological function of the risk variant was further evaluated by performing laboratory experiments. Results Breast cancer risk was significantly associated with three SNPs located at 6q25.1—rs9383935 in CCDC170 and rs2228480 and rs3798758 in ESR1 —with variant allele attributed odds ratios (ORs) of 1.38 (95% confidence interval (CI): 1.20 to 1.57, P  = 2.21 × 10 −6 ), 0.84 (95% CI: 0.72 to 0.98, P  = 0.025) and 1.19 (95% CI: 1.04 to 1.37, P  = 0.013), respectively. The functional variant rs9383935 is in high linkage disequilibrium (LD) with GWAS-reported top-hit SNP (rs2046210), but only rs9383935 showed a strong independent effect in conditional regression analysis. The rs9383935 risk allele A showed decreased activity of reporter gene in both the MCF-7 and BT-474 breast cancer cell lines, which might be due to an altered binding capacity of miR-27a to the 3′ untranslated region (3′ UTR) sequence of CCDC170 . Real-time quantitative reverse transcription PCR confirmed the correlation between rs9383935 genotypes and CCDC170 expression levels. Conclusions The results of this study suggest that the functional variant rs9383935, located at the 3′ UTR of CCDC170 , may be one candidate of the causal variants at 6q25.1 that modulate the risk of breast cancer.