Handwashing with soap effectively reduces exposure to diarrhea-causing pathogens. Interventions to improve hygiene and sanitation conditions in schools within low-income countries have gained increased attention; however, their impact on schoolchildren's exposure to fecal pathogens has not been established. Our trial examined whether a school-based water, sanitation, and hygiene intervention reduced Escherichia coli contamination on pupils' hands in western Kenya. A hygiene promotion and water treatment intervention did not reduce risk of E. coli presence (relative risk [RR] = 0.92, 95% confidence interval [CI] = 0.54-1.56); the addition of new latrines to intervention schools significantly increased risk among girls (RR = 2.63, 95% CI = 1.29-5.34), with a non-significant increase among boys (RR = 1.36, 95% CI = 0.74-2.49). Efforts to increase usage of school latrines by constructing new facilities may pose a risk to children in the absence of sufficient hygiene behavior change, daily provision of soap and water, and anal cleansing materials.