AbstractRotating night shift work is associated with increased risk of breast cancer, likely via circadian disruption. We hypothesized that circadian pathway genes influence breast cancer risk, particularly in rotating night shift workers. We selected 178 common variants across 15 genes pertinent to the circadian system. Using a mixed candidate‐ and tag‐single nucleotide polymorphism approach, we tested for associations between these variants and breast cancer risk in 1,825 women within the Nurses' Health Study II cohort and investigated potential interactions between genotype and rotating shift‐work in a subset of 1,318 women. Multiple‐testing‐adjusted p‐values were obtained by permutation (n = 10,000). None of the selected variants was significantly associated with breast cancer risk. However, when accounting for potential effect modification, rs23051560 (Ala394Thr) in the largest circadian gene, Neuronal PAS domain protein 2 (NPAS2) was most strongly associated with breast cancer risk (nominal test for interaction p‐value = 0.0005; 10,000‐permutation‐based main‐effects p‐value among women with < 24 months of shift‐work = 0.003). The observed multiplicative association with breast cancer risk per minor allele (A) was 0.65 (95% CI = 0.51–0.82) among women with < 24 months of shift‐work and 1.19 (95% CI = 0.93–1.54) with ≥24 months of shift‐work. Women homozygous for the minor allele (AA) with ≥24 months of shift‐work had a 2.83‐times higher breast cancer risk compared to homozygous AA women with <24 months of shift‐work (95% CI = 1.47–5.56). In summary, common variation in circadian genes plays at most a small role in breast cancer risk among women of European ancestry. The impact of NPAS2 Ala394Thr in the presence of rotating shift‐work requires further investigation.