Telomeres are specialized chromatin structures essential for the maintenance of chromosomal integrity and stability. Telomere shortening has been linked to multiple aging-related diseases, including cancer. Evidence associating telomere length with breast cancer risk—most of which has been from retrospective case-control studies—is conflicting. We conducted a nested case-control study based on the Shanghai Women's Health Study (1997–2009) in which we evaluated the association of telomere length and breast cancer risk using peripheral blood samples collected before cancer diagnosis (601 cases and 695 controls). We used monochrome multiplex quantitative polymerase chain reaction to measure relative telomere length. Multiple logistic regressions were used to derive adjusted odds ratios with 95% confidence intervals as the measure of association. Telomere length was inversely correlated with age (r = −0.22). Women with moderately long telomeres (those in the fourth quintile) had the lowest breast cancer risk. Risk increased in a dose-response manner with decreasing quintile of telomere length; odds ratios were 1.39 (95% confidence interval (CI): 0.95, 2.04), 1.79 (95% CI: 1.17, 2.75), and 2.39 (95% CI: 1.45, 3.92), respectively, for the third, second, and first quintiles compared with the fourth quintile. A slightly elevated risk of breast cancer (odds ratio = 1.35, 95% CI: 0.90, 2.04), although one that was not statistically significant, was found in the top quintile (longest telomeres). Our results support the hypothesis that telomere shortening is associated with increased risk of breast cancer and suggest a possible elevated risk associated with long telomeres.