Experimental, epidemiological and clinical studies suggest that calcium and/or its regulating hormones affect breast cancer risk. There has been no prospective cohort study investigating serum calcium levels and breast cancer aggressiveness, as determined by tumour histology and stage. Dichotomized prediagnostic serum calcium levels were investigated in relation to breast cancer aggressiveness as determined by grade (mitotic frequency, tubule formation, nuclear atypia) and stage (tumour size and axillary lymph node status). Cox's proportional hazards analysis and heterogeneity analysis were used to investigate the associations between low/high calcium and grade/stage in a prospective cohort study of 7847 women, out of whom 462 women were diagnosed with incident breast cancer during a mean follow-up of 17.2 years. All analyses were stratified for body mass index and menopausal status. Prediagnostic serum calcium levels in premenopausal women were positively associated with increased tumour aggressiveness as determined by a higher risk of nodal metastasis; relative risk (RR) for calcium above median as compared with calcium below median was 1.88 with a 95% confidence interval (CI) of 1.04-3.38. In overweight women, prediagnostic serum calcium levels were also associated with tumour aggressiveness, as determined by both a higher risk of nodal metastasis [RR (95% CI) 1.69 (0.95-3.02)] and severe nuclear atypia [RR (95% CI) 2.06 (1.10-3.86)]. Results also indicate that, in overweight women, calcium is positively associated with worse grade as determined by tubule formation and mitotic frequency. In conclusion, prediagnostic serum calcium levels are positively associated with increased tumour aggressiveness in premenopausal and/or overweight women.