Arsenic, ubiquitously present as a hazardous metalloid, constitutes a significant danger to human health. Although investigations into the detrimental effects of arsenic on the thyroid have been initiated by researchers, a comprehensive exploration of its toxicological impact and underlying molecular mechanisms remains to be undertaken. In this study, we observed a cumulative effect of arsenic on the thyroid, identifying histological impairments and dysfunction in the thyroid of rats with chronic sodium arsenite (NaAsO2) exposure. These findings indicate that NaAsO2 induces significant thyroid damage. Subsequently, we employed proteomic and phosphoproteomic analyses to investigate the molecular mechanisms underlying the effects of chronic NaAsO2 exposure on the thyroid in rats. We observed that NaAsO2 disrupted the synthesis of thyroid hormones (THs), altered the expression level of THs synthesizing enzyme DUOX2. Additionally, Oxidative phosphorylation, AMPK signaling pathway, Central carbon metabolism in cancer, Cysteine and methionine metabolism, Cellular response to heat stress, and Protein processing in endoplasmic reticulum were upregulated, while Glutathione metabolism was downregulated. In conclusion, this study has unveiled the thyroid damage in rats induced by chronic NaAsO2 exposure and elucidated the disrupted molecular pathways, thereby providing novel insights into the molecular mechanisms underlying arsenic exposure and its impact on thyroid function.