On 10 September 2011, a patient in his 50s was admitted to hospital in Ancona, Italy, after six days of high fever and no response to antibiotics. West Nile virus (WNV) infection was suspected after tests to determine the aetiology of the fever were inconclusive. On 20 September, WNV-specific IgM and IgG antibodies were detected in the patient’s serum. Genomic sequencing of the viral isolate showed that the virus belonged to WNV lineage 2.