Background: Graves' orbitopathy (GO) is a sight-threatening disease associated with thyroid dysfunction, with an unmet medical need for early diagnosis and treatment. Orbital fibroblasts (OFs) proliferation and migration play central roles in the pathogenesis of GO. Homocysteine (Hcy) has been demonstrated to be related to thyroid function, but its role in GO remains unclear. In this study, we aimed to investigate the role of Hcy in GO progression and its effects on OFs. Methods: A total of 131 patients with Graves' disease (GD) were enrolled, of which 68 suffered from GO, with 40 having inactive GO and 28 having active GO. Serum Hcy levels were measured using ELISA assays. Primary cultured OFs were established from orbital connective tissues. Cell proliferation was quantified using CCK-8 assays, while wound healing assays were used to evaluate OFs migration. Western blotting was used to measure Akt signaling pathway. Results: Under a matched thyroid function state, serum Hcy levels were significantly higher in GO patients than in GD. More interestingly, active GO patients had significantly elevated Hcy levels compared to inactive GO patients. Furthermore, serum Hcy levels correlated positively with clinical activity scores in GO patients. In vitro, Hcy stimulated OFs proliferation and migration under both physiological and inflammatory conditions. Mechanistically, Hcy activated the Akt signaling pathway in OFs under these conditions. Conclusions: This study supports the potential role of Hcy as a novel biomarker for GO progression. Furthermore, Hcy stimulates OFs proliferation and migration, suggesting its potential as a therapeutic target for GO treatment.