Approximately 20% cases of familial amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Recent studies have shown that methylene blue (MB) was efficient in conferring protection in several neurological disorders. MB was found to improve mitochondrial function, to reduce reactive oxygen species, to clear aggregates of toxic proteins, and to act as a nitric oxide synthase inhibitor. These pleiotropic effects of relevance to ALS pathogenesis led us to test MB in two models of ALS, SOD1^G93A mice and TDP-43^G348C transgenic mice. Intraperitoneal administration of MB at two different doses was initiated at the beginning of disease onset, at 90 days of age in SOD1^G93A and at 6 months of age in TDP-43^G348C mice. Despite its established neuroprotective properties, MB failed to confer protection in both mouse models of ALS. The lifespan of SOD1^G93A mice was not affected by MB treatment. The declines in motor function, reflex score, and body weight of SOD1^G93A mice remained unchanged. MB treatment had no effect on motor neuron loss and aggregation or misfolding of SOD1. A combination of MB with lithium also failed to provide benefits in SOD1^G93A mice. In TDP-43^G348C mice, MB failed to improve motor function. Cytosolic translocation of TDP-43, ubiquitination and inflammation remained also unchanged after MB treatment of TDP-43^G348C mice.