Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder due to mutations in the SMN1 gene, leading to loss of spinal cord α-motor neurons and proximal muscle weakness. Intravenous administration of self-complementary (sc) recombinant AAV9-hSMN1 vectors have shown efficacy in treating type I SMA patients. However, since scAAV vectors display some limitations, such as limited packaging capacity, we evaluated the therapeutic efficacy of single stranded rAAV9 vectors in the Smn2B/- mouse model. Considering that rAAV vector dosage and transgene expression correlates with therapeutic efficacy, we generated four constructs expressing the human SMN1 coding sequence under various promoters and regulatory sequences. Administration of ssAAV9-hSMN1 vectors at 8e12 vg/kg into the cerebrospinal fluid (CSF) at birth prolonged the lifespan of mutant mice, increased the number of spinal motor neurons and improved the morphology of neuromuscular junctions (NMJ). Systemic administration of two selected vectors was also efficacious in rescuing the phenotype of Smn2B/- mice with survival prolongation, and resulted in SMN protein expression in motor neurons and peripheral tissues. A dose-response study was performed with these vectors in order to determine their minimal effective dose, and showed that overexpression of SMN may also be detrimental in mutant mice. Altogether this work demonstrates the feasibility of SMA gene therapy by administration of ssAAV vectors, which may represent a novel strategy for clinical development. Our results will be discussed in the context of other studies in gene therapy of SMA.