The objective of the present study was to conduct the first systematic review and meta-analysis of prospective studies investigating the associations between total cholesterol (TC), HDL-cholesterol (HDL-C) and LDL-cholesterol (LDL-C) levels and the risk of breast cancer. Relevant studies were identified in PubMed (up to January 2014). Inclusion criteria were original peer-reviewed publications with a prospective design. Random-effects models were used to estimate summary hazard ratios (HR) and 95% CI. Distinction was made between studies that did or did not exclude cancer cases diagnosed during the first years of follow-up, thereby eliminating potential preclinical bias. Overall, the summary HR for the association between TC and breast cancer risk was 0.97 (95% CI 0.94, 1.00; dose-response per 1 mmol/l increment, thirteen studies), and that between HDL-C and breast cancer risk was 0.86 (95% CI 0.69, 1.09; dose-response per 1 mmol/l increment, six studies), with high heterogeneity (I2= 67 and 47%, respectively). For studies that eliminated preclinical bias, an inverse association was observed between the risk of breast cancer and TC (dose-response HR 0.94 (95% CI 0.89, 0.99), seven studies, I2= 78%; highest v. lowest HR 0.82 (95% CI 0.66, 1.02), nine studies, I2= 81%) and HDL-C (dose-response HR 0.81 (95% CI 0.65, 1.02), five studies, I2= 30 %; highest v. lowest HR 0.82 (95% CI 0.69, 0.98), five studies, I2= 0%). There was no association observed between LDL-C and the risk of breast cancer (four studies). The present meta-analysis confirms the evidence of a modest but statistically significant inverse association between TC and more specifically HDL-C and the risk of breast cancer, supported by mechanistic plausibility from experimental studies. Further large prospective studies that adequately control for preclinical bias are needed to confirm the results on the role of cholesterol level and its fractions in the aetiology of breast cancer.