This study was undertaken to determine the seroprevalence of West Nile virus (WNV) in human and equine sera in Iran. Blood samples were tested from 300 human samples and 315 equine samples in five geographic zones of north and central parts of Iran between 2010 and 2012. All samples were tested for the immunoglobulin G (IgG) antibody to WNV by using an enzyme-linked immunosorbent assay (ELISA). Of all samples, 4 (1.3%) human and 9 (2.8%) equines were considered to be seropositive for WNV. These results suggest circulation and exposure of the human and equine populations to WNV in Iran.