Bioengineering of the factor VIII (FVIII) molecule has led to the production of variants that overcome poor secretion and/or rapid inactivation. We tested six modified FVIII variants for in vivo efficacy by expressing them from helper-dependent adenoviral (HD-Ad) vectors. We constructed a wild-type (WT) variant, a B-domain-deleted (BDD) variant, a point mutant for improved secretion (F309S), a variant with a partial B-domain deletion for improved secretion (N6), a combination of the point mutant and partial BDD variant (F309N6), and an inactivation-resistant (IR8) FVIII variant. All the constructs expressed functional protein after injection of high-dose HD-Ad. Activity ranged from 20 to 50% with WT, to approximately 100% with the N6 and F309N6 variants. Interestingly, mice treated with N6 showed long-term FVIII activity and phenotypic correction for up to 74 weeks, with low anti-FVIII antibody titer. Importantly, the N6 variant was therapeutically efficacious even after a 50% reduction of viral dose, thereby indicating that transgene modification itself can improve the dose efficacy of HD-Ad. This finding is significant, because dose efficacy is a key factor in clinical application. In summary, bioengineering of the FVIII molecule may be an effective approach to improving the safety, immunogenicity, and efficacy of HD-Ad gene therapy in hemophilia A (HA).