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Universal gloving may help prevent healthcare-associated infections.However, hand hygiene remains important, even in universal gloving settings.We evaluated whether hand hygiene and gloving were related.Hand hygiene exhibited indication-specific increases after education sessions.Direct observation with feedback and education may help improve compliance.The use of gloves for every patient contact (ie, universal gloving) has been suggested as an infection prevention adjunct and alternative to contact precautions. However, gloves may carry organisms unless they are changed properly. In addition, hand hygiene is required before donning and after removing gloves, and there are scarce data regarding glove changing and hand hygiene in a universal gloving setting.This nonrandomized observational before-after study evaluated the effect of education and feedback regarding hand hygiene. Compliance with hand hygiene and glove use was directly observed in a universal gloving setting at a 10-bed intensive care unit in a Japanese tertiary care university teaching hospital.A total of 6,050 hand hygiene opportunities were identified. Overall, hand hygiene compliance steadily increased from study period 1 (16.1%) to period 5 (56.8%), although there were indication-specific differences in the baseline compliance, the degree of improvement, and the reasons for noncompliance. There were decreases in the compliance with universal gloving and the incidence of methicillin-resistant Staphylococcus aureus.It is difficult to properly perform glove use and hand hygiene in a universal gloving setting, given its complexity. Direct observation with specific feedback and education may be effective in improving compliance.