Our aim was to assess, systematically, the effect of flossing on interproximal caries risk. Six trials involving 808 subjects, ages 4 to 13 years, were identified. There were significant study-to-study differences and a moderate to large potential for bias. Professional flossing performed on school days for 1.7 years on predominantly primary teeth in children was associated with a 40% caries risk reduction (relative risk, 0.60; 95% confidence interval, 0.48-0.76; p-value, < 0.001). Both three-monthly professional flossing for 3 years (relative risk, 0.93; 95% confidence interval, 0.73-1.19; p-value, 0.32) and self-performed flossing in young adolescents for 2 years (relative risk, 1.01; 95% confidence interval, 0.85-1.20; p-value, 0.93) did not reduce caries risk. No flossing trials in adults or under unsupervised conditions could be identified. Professional flossing in children with low fluoride exposures is highly effective in reducing interproximal caries risk. These findings should be extrapolated to more typical floss-users with care, since self-flossing has failed to show an effect.