Lentiviral vectors (LVs) are promising delivery systems for gene therapy. To enhance the efficiency of target cell transduction by LVs, protocols often include the addition of culture additives. In this study, the cationic amphipathic peptide LAH4-L1 (KKALLAHALHLLALLALHLAHALKKA), a DNA transfection agent, was evaluated for its capacity to improve LV infectivity in cell lines and primary cells. Results show that LAH4-L1 enhances infectivity of all LV pseudotypes tested, particularly GALVTR-LVs. More importantly, LAH4-L1 promotes the transduction of CD34+ hematopoietic stem cells with GALVTR-LVs as efficiently as Retronectin, a culture additive used in ex vivo clinical protocols involving LVs. The action of LAH4-L1 relies both on the GALVTR-LV adhesion and post-adhesion steps. LAH4-L1 represents a new and attractive transduction enhancer for hematopoietic gene therapy protocols.