OBJECTIVE: To estimate the global prevalence of handwashing with soap and derive a pooled estimate of the effect of hygiene on diarrhoeal diseases, based on a systematic search of the literature. METHODS: Studies with data on observed rates of handwashing with soap published between 1990 and August 2013 were identified from a systematic search of PubMed, Embase and ISI Web of Knowledge. A separate search was conducted for studies on the effect of hygiene on diarrhoeal disease that included randomised controlled trials, quasi-randomised trials with control group, observational studies using matching techniques and observational studies with a control group where the intervention was well defined. The search used Cochrane Library, Global Health, BIOSIS, PubMed, and Embase databases supplemented with reference lists from previously published systematic reviews to identify studies published between 1970 and August 2013. Results were combined using multilevel modelling for handwashing prevalence and meta-regression for risk estimates. RESULTS: From the 42 studies reporting handwashing prevalence we estimate that approximately 19% of the world population washes hands with soap after contact with excreta (i.e. use of a sanitation facility or contact with children's excreta). Meta-regression of risk estimates suggests that handwashing reduces the risk of diarrhoeal disease by 40% (risk ratio 0.60, 95% CI 0.53-0.68); however, when we included an adjustment for unblinded studies, the effect estimate was reduced to 23% (risk ratio 0.77, 95% CI 0.32-1.86). CONCLUSIONS: Our results show that handwashing after contact with excreta is poorly practiced globally, despite the likely positive health benefits.