Exposure to drinking water disinfection byproducts (DBPs) is potentially associated with adverse developmental effects. Iodoacetic acid (IAA), an unregulated DBP, has been shown to be cytotoxic, mutagenic, genotoxic, and tumorigenic. However, its endocrine-disrupting effects remain unknown. This study evaluated the IAA-induced disruption of the thyroid endocrine system using in vitro and in vivo assays. Rat pituitary tumor GH3 cells were treated with IAA in the presence and absence of triiodothyronine (T3). IAA exposure significantly reduced T3-activated GH3 cell proliferation, indicating the antagonistic activity of IAA in vitro. Sprague-Dawley rats were also subjected to IAA treatment through oral gavage for 28 consecutive days. IAA exposure significantly down-regulated the mRNA expression levels of the thyrotropin receptor (TSHR), the sodium/iodide symporter (NIS), and type I deiodinase and simultaneously reduced the protein expression levels of TSHR and NIS. IAA exposure decreased T3 levels but increased the weights of hypothalamus and the levels of thyrotropin releasing hormone and thyrotropin. In addition, IAA induced the formation of smaller and more depleted follicles or even vacuolization in the thyroid. These results suggested that IAA potentially disrupts the thyroid endocrine system both in vitro and in vivo.