In 2009, six new human cases of West Nile neuroinvasive disease (WNND) were identified in Veneto region, following the six cases already reported in 2008. A human West Nile virus (WNV) isolate was obtained for the first time from an asymptomatic blood donor. Whole genome sequence of the human WNV isolate showed close phylogenetic relatedness to the Italy-1998-WNV strain and to other WNV strains recently isolated in Europe, with the new acquisition of the NS3-Thr249Pro mutation, a trait associated with avian virulence, increased virus transmission, and the occurrence of outbreaks in humans.