Background The mechanism driving dysthyroid optic neuropathy (DON) is unclear. Diffusion-tensor imaging (DTI) allows for noninvasively assessing the microstructure of the entire visual pathway and may facilitate a better understanding of the mechanism of DON. Purpose To assess microstructural changes of the whole visual pathway and to investigate the potential mechanism of trans-synaptic damage(TSD? pathogenesis in DON with DTI. Study Type Cross-sectional. Population Sixty-four patients with bilateral thyroid-associated ophthalmopathy (TAO), 30 with and 34 without DON, and 30 age- and sex-matched healthy controls (HCs). Field Strength/Sequence 3 T/DTI (A single-shot diffusion-weighted echo-planar imaging sequence). Assessment Differences in DTI parameters including fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) in each segment (optic nerve, tract, and radiation) of the entire visual pathway among the groups were compared. The parameters of visual evoked potentials (VEPs), visual field tests, and mean retinal nerve fiber layer (mRNFL) thickness on optical coherence tomography were also compared across patients. Statistical Tests Student's t-test, chi-square test; ANOVA with post-hoc testing, interclass correlation coefficient, and correlation analysis. Significance level: P < 0.05. Results TAO patients with DON showed significantly reduced mRNFL thickness and abnormal VEPs. There was a tendency for gradually reduced FA and AD, and increased RD and MD from HCs, with non-DON to with DON in optic nerve and tract, statistically. For radiation, the RD and MD showed statistical increase, the AD and FA just showed numerical decrease (P = 0.119 and 0.059, respectively). For DON, the FA and MD of visual pathway segments showed correlations with abnormal VEPs. Data Conclusion DTI may be a useful tool for detecting microstructural changes in the entire visual pathway in DON. The changes in RNFL thickness and DTI parameters suggested TSD as a potential pathogenic mechanism of DON. Evidence Level: 4 Technical Efficacy: Stage 5