Fibrosis is the late stage of thyroid-associated ophthalmopathy (TAO), resulting in serious complications. Effective therapeutic drugs are still lacking. We aimed to explore the mechanism of TAO fibrosis and to find a targeted drug. High-throughput RNA sequencing was performed on orbital connective tissues from twelve patients with TAO and six healthy controls. Protein–protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) database and we identified the hub gene by Cytoscape software. Additionally, the RNA sequencing results were validated by quantitative real-time polymerase chain reaction (qRT–PCR). Bioinformatic prediction identified the functions of differentially expressed genes (DEGs). Further orbital connective tissue and serum samples of the TAO and control groups were collected for subsequent experiments. Histologic staining, Western blotting (WB), qRT–PCR, enzyme-linked immunosorbent assays (ELISAs), gene overexpression through lentiviral infection or silencing gene by short interfering RNA (siRNA) were performed. We found that the relaxin signaling pathway is an important regulatory pathway in TAO fibrosis pathogenesis. Serelaxin exerts antifibrotic and anti-inflammatory effects in TAO. Furthermore, the downstream Notch pathway was activated by serelaxin and was essential to the antifibrotic effect of serelaxin in TAO. The antifibrotic effect of serelaxin is dependent on RXFP1.