Purpose This study was designed to investigate whether transduction of lentiviral vectors (LV) carrying human coagulation factor VIII (hFVIII) cDNA into skeletal muscle could increase circulating hFVIII concentrations. Materials and Methods A LV containing bacterial LacZ gene as a control or human FVIII gene was intramuscularly administered into the thigh muscle of 5 weeks old Sparague-Dawley rats. The plasma human FVIII concentration and neutralizing anti-FVIII antibodies were measured for up to 12 weeks in these experimental animals. Results The plasma human FVIII levels in the rats injected with LV carrying FVIII cDNA peaked at post-injection 1st week (5.19 ± 0.14 ng/mL vs. 0.21 ± 0.05 ng/mL in control rats , p < 0.05). Elevated hFVIII concentrations were maintained for 4 weeks (2.52 ± 0.83 ng/mL vs. 0.17 ± 0.08 ng/mL in control rats, p < 0.05) after a single intramuscular injection. In the Bethesda assay, neutralizing antibodies for FVIII protein were detected only in FVIII-LV injected rats by the 10th week, but not in control rats. Conclusion This study suggested that a single administration of an advanced generation LV carrying the human FVIII cDNA resulted in elevation of FVIII level in immune competent rats, and that this gene transfer approach to the skeletal muscle could be an effective tool in treatment of hemophilia A.