Chlorpyrifos (CPF) is commonly used for agricultural and domestic applications, and its contamination is widely detected in environmental media, and in a broad spectrum of field crops, fruits and vegetables. CPF exposure causes many adverse effects on human health including hepatoxicity, neurotoxicity and endocrine disruption. However, few studies have been conducted thus far to investigate the potential influence of CPF exposure on iron metabolism at concentrations that do not trigger significant cell death. Iron metabolism is concertedly governed by the hepcidin-ferroportin axis, where hepcidin is the central hormone involved in the regulation of iron absorption and release, while ferroportin is the only known iron exporter that functions by iron egress from cells. In the present study, we demonstrated that CPF treatment at a non-toxic concentration greatly enhanced ferroportin gene transcription in human macrophage THP-1 cells. CPF significantly inhibited hepcidin expression in human hepatocyte HepG2 cells. As a result, the intracellular labile iron pool (LIP) was largely reduced in THP-1 and HepG2 cells. The combined data of this study identified a novel finding of CPF that disrupts iron homeostasis by altering ferroportin and hepcidin expression. These findings would be useful in understanding the biological effects of CPF exposure, especially under relatively low and non-toxic doses.