Human Adenovirus type 5 (Ad5) has been extensively explored in clinical gene therapy, but its immunogenicity dramatically affects the kinetics and toxicity profile of the vector. We previously designed a variety of artificial lipid bilayer envelopes around the viral capsid to develop safer hybrid vectors. Here, we studied the interaction of enveloped Ad in cationic (DOTAP:Chol) or anionic (DOPE:CHEMS) lipid bilayers with different blood components. When Ad was enveloped by cationic lipids, significantly high levels of viral uptake in HepG2 cultured cells were achieved, independent of blood coagulation factors present. In vitroexperiments also showed that artificial envelopment of Ad completely altered the affinity towards both human and murine red blood cells. After intravenous administration in BALB/c mice, real-time PCR and transgene expression studies indicated that cationic lipid envelopes significantly reduced hepatocyte transduction significantly increasing virus lung accumulation compared to DOPE:CHEMS enveloped or naked Ad. ALT/AST serum levels and liver histology showed that envelopment also improved hepatotoxicity profiles compared to naked Ad. This study suggests that artificial envelopes for Ad significantly alter the interactions with blood components and can divert viral particles from their natural liver tropism resulting in reduced hepatotoxicity.