Retinal Müller glial cells have been shown to undergo reactive gliosis in a variety of retinal diseases. Upregulation of glial fibrillary acidic protein (GFAP) is a hallmark of Müller cell activation. Reactive gliosis after retinal detachment or ischemia/reperfusion is characterized by hypertrophy and downregulation of inwardly rectifying K+ (Kir) currents. However, this kind of physiological alteration could not be detected in slowly progressing retinal degenerations. The photoreceptor toxin N-methyl-N-nitrosourea (MNU) leads to the rapid loss of cells in the outer nuclear layer and subsequent Müller cell activation. Here, we investigated whether Müller cells from MNU-treated mice exhibit reactive gliosis. We found that Müller cells showed increased GFAP expression and increased membrane capacitance, indicating hypertrophy. Membrane potential and Kir channel-mediated K+ currents were not significantly altered whereas Kir4.1 mRNA expression and Kir-mediated inward current densities were markedly decreased. This suggests that MNU-induced Müller cell gliosis is characterized by plasma membrane increase without alteration in the membrane content of Kir channels. Taken together, our findings show that Müller cells of MNU-treated mice are reactive and respond with a form of gliosis which is characterized by cellular hypertrophy but no changes in Kir current amplitudes.