The aim of present study was to assess the effects of a eutectic mixture of topical anesthetics (EMLA cream) on pain responses of preschoolers during venipuncture. The pain responses were reported by children, nurses, and the children’s mothers. The pre- and post-test-designed study, which included a non-equivalent control group, was carried out in a pediatric unit at E University Hospital in South Korea. Pain responses during venipuncture were measured by the self-reporting of the children using the Face Pain Rating Scale (FPRS), the Procedure Behavior Checklist by nurses (PBCL), the Visual Analog Scale (VAS) by mothers, in addition to measurements of the children’s pulse rate and level of oxygen saturation. EMLA cream was effective in decreasing the pain responses of the children as assessed by FPRS, PBCL, and VAS scores. However, pulse rate and level of oxygen saturation of the children during venipuncture were not significantly different between the two groups. EMLA cream could be a useful option for reducing pain in preschool children during invasive procedures.