Zinc oxide (ZnO) works as a long-lasting, broad-spectrum physical sunblock, and can prevent skin cancer, sunburn, and photoaging. Nanosized ZnO particles are used often in sunscreens due to consumer preference over larger sizes, which appear opaque when dermally applied. Although the US Food and Drug Administration approved the use of nanoparticles (NPs) in sunscreens in 1999, there are ongoing safety concerns. The aim of this study was to evaluate the subchronic toxicity of ZnO NPs after dermal application according to the Organization for Economic Cooperation and Development Test Guidelines 411 using Good Laboratory Practice. Sprague Dawley rats were randomly divided into eight (one control, one vehicle control, three experimental, and three recovery) groups. Different concentrations of ZnO NPs were dermally applied to the rats in the experimental groups for 90 days. Clinical observations as well as weight and food consumption were measured and recorded daily. Hematology and biochemistry parameters were determined. Gross pathologic and histopathologic examinations were performed on selected tissues from all animals. Analyses of tissue were undertaken to determine target organ tissue distribution. There was no increased mortality in the experimental group. Although there was dose-dependent irritation at the site of application, there were no abnormal findings related to ZnO NPs in other organs. Increased concentrations of ZnO in the liver, small intestine, large intestine, and feces were thought to result from oral ingestion of ZnO NPs via licking. Penetration of ZnO NPs through the skin seemed to be limited via the dermal route. This study demonstrates that there was no observed adverse effect of ZnO NPs up to 1,000 mg/kg body weight when they are applied dermally.