Pathological neuron-glia interactions contribute to the progress of ALS. Astroglial activation encloses rapid morphological reactions and leads to microglial inflammation. Pro-inflammatory microglia as the other determinants of disease progression are essentially involved in the noncell-autonomous motoneuronal degeneration during symptomatic stages. Methylene blue (MB) inhibits the effect of nitric oxide, which mediates microglial response to injuries. Using ALS-linked transgenic SOD1G93A mice containing labeled projection neurons and microglia, in vivo 2P-LSM imaging was used to investigate the effect of MB on microglia-mediated inflammation in affected motor parts of the spinal cord. In contrast to local superfusion of the lateral spinal cord with MB, systemically treatment led to no alteration of microglial reaction towards laser-induced axon transection in SOD1G93A mice. Oral and intraperitoneal MB treatment of SOD1G93A mice delayed disease onset, but not progression of disease or the survival rate. Accordingly, the number of motor neurons was higher in late-presymptomatic stage by systemically applied MB while microgliosis was unchanged. This data suggest a critical influence of systemically applied MB on mutated motor neurons while mutated microglia are unaltered. Thus, the beneficial effect of systemic MB administration on mSOD1 disease onset seems to be independent of microglia-mediated inflammation.