West Nile virus (WNV) is a mosquito-borne virus belonging to the family Flaviviridae included in the Japanese encephalitis antigenic complex (JEAC). A seroepidemiological study was carried out in 2011 using 160 horse sera collected from different areas of Bolivia to investigate the presence of WNV antibody. A high proportion (59.4%) of the tested sera were positive to a commercially available WNV competitive enzyme-linked immunosorbent assay (C-ELISA). Sixty-six randomly selected C-ELISA-positive sera were further tested by WNV plaque reduction neutralization test (PRNT), virus neutralization (VN), and immunoglobulin M (IgM)-WNV ELISA to exclude false-positive results due to possible cross-reactions to other members of the JEAC and to investigate if the horses were recently infected. No WNV IgM was detected in these samples, whereas neutralizing antibodies were found in 21 and 18 samples by PRNT and VN, respectively. In conclusion, a high proportion of the Bolivian horses included in this study reacted serologically against viruses of the JEAC. WNV was partially responsible (31.8%) for these reactions, supporting the conclusion that WNV circulated in Bolivia.