In previous studies, we achieved safe and efficient hematopoietic stem cell (HSC) transduction in mobilized mice and macaques with intravenously injected helper-dependent adenovirus HDAd5/35++ vectors. These vectors are derivatives of serotype Ad5-containing CD46-affinity enhanced Ad35 fiber knob domains. Considering the impact of anti-Ad5/HDAd5/35++ neutralizing serum antibodies present in the human population, we generated HSC-retargeted HDAd6/35++ vectors derived from serotype 6. We found a lower prevalence and titers of serum anti-HDAd6/35++ in human samples compared with HDAd5/35++. HDAd6/35++ vectors efficiently transduced human and rhesus CD34 cells . Intravenous injection of HDAd5/35++-GFP or HDAd6/35++-GFP vectors after G-CSF/AMD3100 mobilization of mice with established human hematopoiesis or human CD46 transgenic mice resulted in comparable GFP marking rates in HSCs in the bone marrow and spleen. In long-term HSC transduction and selection studies with integrating vectors, stable GFP expression in >75% of PBMCs was show for both vectors. In contrast with HDAd5/35++, undesired transduction of hepatocytes was minimal with HDAd6/35++. Furthermore, HDAd6/35++ allowed for efficient HSC transduction in Ad5-pre-immune mice. These features, together with the straightforward production of HDAd6/35++ vectors at high yield, make this new HDAd vector platform attractive for clinical translation of the approach.