Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective death of motoneurons (MNs). Our previous results revealed that intravenous administration of mesenchymal stem cells (MSCs) improved different pathological features in SOD1G93A ALS mice. We hypothesised that the beneficial effect could be mediated by paracrine activity of extracellular vesicles (EVs) secreted by MSCs shuttling microRNAs . Subsequently, we studied in-vitro the activity of MSCs-derived EVs on spinal cord astrocytes of symptomatic SOD1G93A mice. The results demonstrated that 24h in-vitro exposure to MSC-derived EVs was able to reduce the aberrant reactive phenotype of ALS astrocytes, reducing the levels of activation markers and the secretion of pro-inflammatory factors. Most importantly, the neurotoxicity of ALS astrocytes towards co-cultured MNs was significantly reduced after the exposure to MSCs-derived EVs. Interestingly, the transfection with synthetic mimics of miRNAs, found upregulated in MSCs, reverted the reactive phenotype of SOD1G93A ALS astrocytes. As translational step, we evaluated in-vivo the effects of chronic intranasal administration of MSC-derived EVs in SOD1G93A mice by performing behavioural studies and ex-vivo analyses. Our results demonstrated that MSC-derived EVs efficiently get into the CNS and ameliorate motor functions scores and the survival probability of SOD1G93A mice. Overall, our data pave the way for the application of MSCs-derived EVs or even EV-mimicking synthetic particles as an innovative and promising therapeutic strategy for ALS cure.