BACKGROUND: Tributyltin (TBT), a prevalent environmental antiseptic, contaminates seafood, fish, and drinking water, posing health risks. While TBT's hepatic toxicity is well-known, its sex-specific effects on liver function remain poorly understood. METHODS: To address this gap, a comprehensive analysis was conducted utilizing the Toxicant Exposures and Responses by Genomic and Epigenomic Regulators of Transcription (TaRGET) dataset. Chromatin accessibility changes and transcriptomic alterations were analyzed via ATAC-seq and RNA-seq in liver tissues from TBT-exposed male and female mice. In vitro experiments were performed to validate the key bioinformatic findings. RESULTS: TBT exposure induced significant chromatin accessibility changes and transcriptomic alterations in male liver compared to female counterparts. Notably, Signal transducer and activator of transcription 3 (STAT3) was identified as a central regulator among differentially expressed genes (DEGs) in male liver cells. Functional validation experiments confirmed that TBT-mediated downregulation of STAT3 impaired liver cell function and contributed to increased hepatotoxicity in males. CONCLUSIONS: Our study highlights significant sex-dependent differences in TBT-induced hepatotoxicity and identifies STAT3 as a critical mediator in male liver cells, providing a novel perspective on the toxicology of TBT.