The main purpose of this work was to evaluate the transfection of novel DNA vectors, minicircles (mC), on embryonic stem cell-derived neural stem cells (NSC). We demonstrated that by combining microporation with mC, 75% of NSC expressing a transgene is achieved without compromising cell survival, morphology, and differentiation potential. When comparing mC with their plasmid DNA (pDNA) counterparts, both gave rise to similar transfection levels but cells harboring mC showed 10% higher cell viability, maintaining 90% of survival at least for 10 days. Long-term analysis showed that NSC harbor a higher number of mC copies and consequently exhibit higher transgene expression when compared to their pDNA counterpart. Taken together, our results offer the first insights on the use of mC as a novel and safe strategy to genetically engineer NSC envisaging their use as biopharmaceuticals in clinical settings for the treatment of neurodegenerative or neurological diseases.