It has been suggested that women living in urban areas are at higher risk of breast cancer (BC) compared to women living in rural areas. However, most published studies on this topic are ecological and did not adjust for individual BC risk factors. We investigated this hypothesis in a French prospective cohort. Cox proportional hazards regression models were used to evaluate the association between birth or residence in an urban area and BC risk among 75,889 women of the French E3N cohort (aged 38-66 years at recruitment in 1990) before and after adjustment for known BC risk factors and stratifying on birth cohort. From 1990 to 2008, a total of 5,145 BC cases were diagnosed. Being born in an urban area was associated with BC risk before (HR 1.11, 95% CI 1.05-1.18) and after (HR 1.07, 95% CI 1.01-1.14) adjustment for known BC risk factors. Living in an urban area in 1990 was not associated with BC risk. Being born in an urban area is associated with a weak increase in BC risk. This may be suggestive of higher exposure to air pollution and to other environmental exposures, to be investigated in future studies.