Azoxystrobin is a broad-spectrum fungicide having a wide usage. However, the toxic effect of azoxystrobin in humans is not reported. In Japan, azoxystrobin was detected at a five-fold higher concentration than the normal upper limit (2.5 mg/kg) in a shipment of an Australian barley used in different food products. Thus, there is a chance of azoxystrobin exposure through food to humans, and hence it is imperative to study the toxic effects of this compound. In this study, the toxic effect of azoxystrobin was evaluated to predict its adverse effects on human. Azoxystrobin at 3-30 µM (approximately 1.2-12.1 mg/L) raised the intracellular Zn²⁺ concentration of rat thymic lymphocytes. This increase was due to an influx of extracellular Zn²⁺ and a release of intracellular Zn²⁺. Azoxystrobin partially inhibited the temperature-dependent Zn²⁺ influx, thus jeopardizing the cellular Zn²⁺ homeostasis. Because Zn²⁺ is an important intracellular messenger in lymphocytes, this altered Zn²⁺ homeostasis might lead to adverse effects if the blood concentration of azoxystrobin reaches 3 µM or more in humans. However, by extrapolating the azoxystrobin pharmacokinetics data of rats to human, it can be predicted that such high blood concentration may be unlikely in humans.