Arsenic, an environmental toxicant, causes various human diseases. Recently, a few of studies have reported that arsenic has the potential to negatively affect the endocrine system and induce thyroid toxicity, but its underlying mechanisms remain unclear. Here, we sought to observe thyrotoxicity of sodium arsenite (NaAsO₂), and explore the role of the phosphatidylinositol 3-kinase (PI3K) and transcription factor NF-E2-related factor 2 (Nrf2) signalling process. Wistar rats were treated with 0, 0.8, 4.0, and 20.0 mg/kg NaAsO₂ for 19 weeks. Our results revealed that NaAsO₂ exposure resulted in histopathological changes of thyroid in rats, which accompanied by the decreased serum T3, T4 and TG levels and the increased serum E2 level. ERα and TRα mRNA expression decreased in the genomes of rats exposed to low levels of arsenic. Keap1, Nrf2, PI3K and AKT mRNA expression levels were elevated in the arsenic-exposed group. The NaAsO₂-exposed groups showed significant increases in the PI3K, AKT and Nrf2 protein levels. Alongside the increase doses of NaAsO₂ exposure group of 0, 0.4, 0.8, 3.2 μM, T3 and T4 levels in Nthy-ori 3-1 cells were also reduced. The ERα and TRα mRNA levels increased significantly, intracellular Keap1 and AKT mRNA expression decreased, while Nrf2 and PI3K mRNA expression increased. Collectively, our findings suggest that NaAsO₂ exposure may induce cytotoxicity and oestrogen production in cells, causes dysfunction of thyroid in Wistar rats, in which the PI3K/Nrf2 pathway may play a significant role.