Abstract Bovine viral diarrhea virus (BVDV), a Flavivirus, plays a key role in bovine respiratory disease complex, which leads to pneumonia and death of calves. Current vaccines are not very effective due, in part, to immunosuppressive traits of the virus. There are diverse BVDV strains and thus, current vaccines contain type 1 and 2 viruses (BVDV-1 & 2). Modified live virus (MLV) vaccines are superior to killed virus, but they are susceptible to antibody neutralization and complement-mediated destruction that limits their efficacy in neonates. We generated three novel mosaic polypeptide chimeras, designated NproE2123; NS231; and NS232, which represent determinants conserved among BVDV strains and these antigens are the main targets involved in protection. We confirmed that a proto-type adenovirus-vectored vaccine expressing these antigens was recognized by mAbs, sera, and T cells raised against diverse BVDV strains. In a poof-of-concept efficacy study, the vaccine induced significantly higher IFN-γ spot forming cells (p<0.05), T cell proliferation (p<0.05), and SN titers (p<0.05) against BVDV-1 & 2 as compared to immune responses induced by a commercial MLV vaccine. Following challenge, the calves immunized with the proto-type and the MLV vaccines had mild clinical scores compared to naïve controls. These results support the hypothesis that a broadly protective subunit vaccine can be generated using mosaic polypeptides that incorporate protective motifs from diverse BVDV strains.