Objectives: In diabetic subjects, retinal neurodegeneration is associated with reactive gliosis which involve the activation of Müller cells (MCs), mainly characterized by increased expression of glial fibrillary acidic protein (GFAP). Recently, in lower vertebrates, it has been reported that, following reactive gliosis, MCs acquire characteristics of retinal stem cells and that Sonic Hedgehog (SHH) is a crucial regulator of this de-differentiation process. Moreover, it has been reported that SRY-Box Transcription Factor 2 (SOX2) is required for the proliferative and differentiation capacity of retinal progenitors. Although diabetic condition causes reactive gliosis in the retina, the effect of glycemic fluctuations on activation and reprogramming of human MCs is yet unknown. The aim of the study is to assess the activation and reprogramming of the human Müller cell line MIO-M1 exposed to high glucose (HG) and glucose variability (GV). Methods: Human Muller cell line MIO-M1 was cultured in a medium containing either 5 mM (N cells) or 25 mM of glucose (H cells) and then incubated for 96 h in a medium with: basal glucose (5 or 25 mM), high glucose (25 or 45 mM), basal glucose and high glucose alternated every 24 hours (5/25 or 25/45 mM), low glucose and high glucose alternated every 24 hours (3/25 or 5/45 mM). MCs activation was studied through the expression of GFAP and MCs reprogramming was studied through the expression of SHH and SOX2, evaluated by western blot analysis. In addition, a morphological analysis of MCs was performed by using GFAP and SHH immunolabelling on micrographs obtained with a fluorescence microscope. Results: In N cells, a significant upregulation of GFAP expression was observed in response to HG and GV conditions (p<0.05). Moreover, this increase was associated with morphological changes and a higher number of hypertrophic cells (p<0.05). In H cells, there was no modulation of GFAP expression in response to either treatments. In N cells, a significant upregulation of SHH and SOX2 was also observed in response to HG and GV treatments, with a peculiar localization of the SHH in multiple spots inside the cytoplasm. On the contrary, H cells showed a reduced expression of SHH and SOX2 (p<0.01) in response to the treatments without morphological changes of SHH localization. Conclusions: Our results highlight activation and reprogramming features of human Muller cell line MIO-M1 cultured in normal glucose and exposed to HG and GV conditions.