Background Breast cancer risk is temporarily increased after a full-term pregnancy and declines thereafter, possibly due to increased levels of gonadal and placental hormones during pregnancy. Inconsistent results, however, have been reported after twin pregnancies with higher hormone levels. Among women treated with in vitro fertilisation (IVF), for whom the number of embryos available for implantation is known, we recently observed that a multiple birth after implantation of all transferred embryos is associated with higher levels of vascular endothelial growth factor (VEGF). As VEGF is involved in breast cancer progression, we studied the effects of embryo implantation and a multiple birth on breast cancer risk in a nationwide Dutch cohort of IVF-treated women. Methods We performed a cohort analysis among 12,589 women who had been treated with IVF between 1983 and 1995 and completed a risk factor questionnaire between 1997 and 1999. Data on IVF treatment were obtained from medical records. Breast cancer cases were ascertained through linkage with the population-based Netherlands Cancer Registry. Breast cancer risks associated with singleton and multiple births were estimated with Cox regression. Findings There were 1688 women (13.4%) with multiples, 6027 (47.9%) with singletons and 4874 (38.7%) nulliparous women. Breast cancer occurred in 317 women of whom 57 had multiples. Breast cancer risk was 1.44 times higher in mothers of multiples than in mothers of singletons (95% confidence interval (CI) 1.06-1.97). Risk was highest in women who gave birth to multiples from all embryos transferred (adjusted hazard ratio (HR) 1.86, 95% CI 1.01-3.43), and lower for those with multiples after incomplete embryo implantation (adjusted HR 1.31, 95% CI 0.76-2.25). Interpretation A woman's potential to implant all transferred embryos may be associated with breast cancer risk. Further research is needed to confirm our results and to identify the underlying biological mechanisms.