Abstract Background Neural stem cell (NSC) transplantation holds promising therapeutic potential for neurodegenerative disorders like amyotrophic lateral sclerosis (ALS). However, pre-clinical studies and early-phase clinical trials have faced challenges hindering the effective clinical translation of this approach. Crucial hurdles include the side-effects of prolonged immunosuppression, concerns regarding cell origin and transplantation dosage, identification of the most appropriate therapeutic window, and invasiveness of surgical procedures. Here, we assessed the safety and efficacy of intracerebroventricular (ICV) hNSC transplantation as a novel and possibly more effective experimental approach for ALS. Methods We evaluated the safety of administering up to 1 × 106 hNSCs in immunodeficient mice and assessed their potential efficacy in reducing ALS hallmarks employing the SOD1G93A mouse model. Both transient (15 days) and prolonged immunosuppression regimens, at low (15 mg/kg) and high (30 mg/kg) doses, were tested along with two different cell dosages (3 × 105 and 1 × 106). Results Our study suggests that: (i) a bilateral ICV transplantation of 1 × 106 hNSCs is safe and non-tumorigenic in immunodeficient hosts; (ii) sustained and high-dose immunosuppression is essential for ensuring cell survival in immunocompetent SOD1G93A mice; and (iii) hNSCs may delay motor symptom progression and reduce spinal cord microgliosis in SOD1G93A mice when administered in the lateral ventricles under prolonged high-dose (30 mg/kg) immunosuppression. Conclusions ICV transplantation of hNSCs emerges as a safe and promising strategy for ALS, demonstrating potential to delay motor decline and reduce spinal cord microgliosis. However, sustained high-dose immunosuppression is crucial for therapeutic efficacy, emphasizing the need for further optimization to overcome translational challenges and achieve durable clinical benefits.