We examined breast cancer risk in association with overall glycemic index (GI), glycemic load (GL), and dietary carbohydrate and sugar intake in a prospective cohort of 49,613 Canadian women enrolled in the National Breast Screening Study who completed a self-administered food frequency questionnaire between 1980 and 1985. Linkages to national mortality and cancer databases yielded data on deaths and cancer incidence, with follow-up ending between 1998 and 2000. During a mean follow-up of 16.6 years, we observed 1,461 incident breast cancer cases. GI, GL, total carbohydrate and total sugar intake were not associated with breast cancer risk in the total cohort. However, there was evidence of effect modification of the association between GI and breast cancer risk by menopausal status (p = 0.01), the hazard ratio for the highest versus the lowest quintile level of GI being 0.78 (95% CI = 0.52-1.16; ptrend = 0.12) in premenopausal women and 1.87 (95% CI = 1.18-2.97; ptrend = 0.01) in postmenopausal women. The associations between GI and GL were not modified by body mass index (BMI) or by vigorous physical activity among pre- or postmenopausal women. Similarly, the associations between GI/GL and risk in postmenopausal women were not modified by BMI, vigorous physical activity, or ever use of hormone replacement therapy (HRT), although the associations were slightly stronger among those who reported no vigorous physical activity (ptrend = 0.02), among those who reported ever using HRT (ptrend = 0.02) and among normal-weight women (BMI < 25 kg/m2; ptrend = 0.03). Our data suggest that consumption of diets with high GI values may be associated with increased risk of breast cancer among postmenopausal women, possibly more so among subgroups defined by participation in vigorous physical activity, ever use of HRT and those who are not overweight.