ABSTRACT Bluetongue virus (BTV) causes bluetongue, a major hemorrhagic disease of ruminants. In order to investigate the molecular determinants of BTV virulence, we used a BTV8 strain minimally passaged in tissue culture (termed BTV8 L in this study) and a derivative strain passaged extensively in tissue culture (BTV8 H ) in in vitro and in vivo studies. BTV8 L was pathogenic in both IFNAR −/− mice and in sheep, while BTV8 H was attenuated in both species. To identify genetic changes which led to BTV8 H attenuation, we generated 34 reassortants between BTV8 L and BTV8 H . We found that partial attenuation of BTV8 L in IFNAR −/− mice was achieved by simply replacing genomic segment 2 (Seg2, encoding VP2) or Seg10 (encoding NS3) with the BTV8 H homologous segments. Fully attenuated viruses required at least two genome segments from BTV8 H , including Seg2 with either Seg1 (encoding VP1), Seg6 (encoding VP6 and NS4), or Seg10 (encoding NS3). Conversely, full reversion of virulence of BTV8 H required at least five genomic segments of BTV8 L . We also demonstrated that BTV8 H acquired an increased affinity for glycosaminoglycan receptors during passaging in cell culture due to mutations in its VP2 protein. Replication of BTV8 H was relatively poor in interferon (IFN)-competent primary ovine endothelial cells compared to replication of BTV8 L , and this phenotype was determined by several viral genomic segments, including Seg4 and Seg9. This study demonstrated that multiple viral proteins contribute to BTV8 virulence. VP2 and NS3 are primary determinants of BTV pathogenesis, but VP1, VP5, VP4, VP6, and VP7 also contribute to virulence. IMPORTANCE Bluetongue is one of the major infectious diseases of ruminants, and it is listed as a notifiable disease by the World Organization for Animal Health (OIE). The clinical outcome of BTV infection varies considerably and depends on environmental and host- and virus-specific factors. Over the years, BTV serotypes/strains with various degrees of virulence (including nonpathogenic strains) have been described in different geographical locations. However, no data are available to correlate the BTV genotype to virulence. This study shows that BTV virulence is determined by different viral genomic segments. The data obtained will help to characterize thoroughly the pathogenesis of bluetongue. The possibility to determine the pathogenicity of virus isolates on the basis of their genome sequences will help in the design of control strategies that fit the risk posed by new emerging BTV strains.