Chronic exposure to arsenic in drinking water is associated with an increased risk of bladder cancer in arseniasis-endemic areas throughout the world. Human epidermal growth factor receptor 2 (HER2) was recently reported to be involved in the development of bladder cancer. However, until this point, little is known about HER2 activation and its mechanism in arsenite-exposed urothelial cells. The aim of this study was to identify factors associated with HER2 activation in an arsenite-exposed human bladder epithelial cell line. Results of this study demonstrated that levels of phosphorylated HER2 increased significantly in cells treated with arsenite. Additionally, the protein levels of epidermal growth factor (EGF), transforming growth factor α (TGFα), soluble ectodomain fragment of E-cadherin (sE-cad), and neuregulin 1 (NRG1) were also increased significantly in these cells. Meanwhile, the protein levels of heat shock protein 90 (HSP90) and plasma membrane calcium ATPases 2 (PMCA2) increased, while those of Interleukin-6 (IL-6) and N-myc downstream regulated gene 1 (NDRG1) decreased significantly. Pretreatment of arsenite-exposed cells with exogenous EGF, TGFα, NRG1, and HSP90 could promote, whereas exogenous IL-6 and NDRG1 could suppress, the phosphorylation of HER2. Furthermore, reduction of EGF, TGFα, NRG1, PMCA2, or HSP90 via its neutralizing antibody, siRNA, or inhibitor suppressed, whereas knockdown of E-cadherin promoted, the phosphorylation of HER2. In conclusion, our results suggested that HER2 might be activated through promoting the dimerization of HER2 with other members of HER family, maintaining the stability of phosphorylated HER2, and attenuating the suppression of HER2 activation in arsenite-exposed cells.