Hematopoietic stem cells (HSC) are quiescent, self-renewing cells that can give rise to all blood cell lineages. HSC are an attractive target for gene therapy, due to their differentiation capacity and the number of diseases that result from abnormal HSC function. While human HSC have been shown to be transduced with adenoviral vectors, the adenoviral transduction of mouse HSC has not been extensively studied. We show here that a population of mouse bone marrow highly enriched for HSC (called side population, or SP, cells) can be transduced with adenovirus type 5 (Ad5) at a low multiplicity of infection. Transduced SP cells showed normal in vitro myeloid differentiation potential compared to mock-transduced SP cells. Transduced SP cells retained substantial but reduced in vivo long-term repopulating activity and contributed to all blood cell lineages. Ad5 transduction of mouse SP cells was dependent on coxsackie and adenovirus receptor (CAR), as an anti-CAR blocking antibody greatly reduced transduction. Therefore, adenoviral transduction of mouse HSC can be achieved without ablation of the hematopoietic repopulating activity.