Abstract Background Fabry disease (FD) is a rare X-linked, lysosomal storage disorder caused by mutations in the alpha-galactosidase gene and characterized by neurological, cutaneous, renal, cardiovascular, cochleo-vestibular and ocular manifestations. The aim of this study is to characterize morphological, functional and autophagy-lysosome pathway alterations of the ocular surface in FD patients. Methods Eleven subjects with a diagnosis of FD and fifteen healthy control subjects were examined. All patients underwent ocular surface slit lamp examination, corneal aesthesiometry and in vivo confocal laser-scanning microscopy (CCM). Conjunctival impression cytology was performed in six FD patients and six controls, to assess for expression of two markers of the autophagy-lysosome pathway: the microtubule-associated protein light chain 3 (LC3) and lysosome-associated membrane protein 2 (LAMP2). Results Cornea verticillata and increased conjunctival vessel tortuosity were detected respectively in 67% and 33% of patients with FD. Compared with healthy subjects, patients affected by FD showed a significant reduction in corneal nerve fiber length, density and nerve branching on CCM and a significantly increased expression of LC3 on conjunctival impression cytology (p