Amyotrophic lateral sclerosis (ALS) is a progressive and devastating neurodegenerative disease caused by selective degeneration and death of motor neurons. So far very limited therapeutic options have emerged to treat this fatal disease. Homocysteine (Hcy) lowering drugs have been suggested to be a palliative therapy of this disease. Folate, Vitamin B12 (VitB12) and Vitamin B6 (VitB6) are important elements involved in the Hcy metabolism and we proposed that medications which could promote the absorption of folate, VitB12 and VitB6 might have benefit for ALS. Galactooligosaccharides (GOS) is a prebiotic which could significantly improve the absorption and syntheses of B Vitamins. To investigate whether GOS could provide neuroprotective effect in ALS, we applied GOS and GOS-rich prebiotic yogurt in SOD1G93A mice and assessed their effects on the disease progression of ALS. Our results showed that GOS and prebiotics yogurt administration significantly delayed the disease onset and prolonged the lifespan in SOD1G93A mice. Also, these products increased the concentration of folate, VitB12 and reduced the level of Hcy. Moreover, we found that both GOS and prebiotics yogurt attenuated motor neurons loss, improved the atrophy and mitochondrial activity in myocyte. Furthermore, we demonstrated that GOS and GOS-rich prebiotic treatment suppressed the activation of astrocytes and microglia and regulated several inflammatory- and apoptosis-related factors. Our findings suggested that GOS might have therapeutic potential for ALS, and GOS-rich prebiotic yogurt might be considered as a nutritional therapy for this disease.