We described the serological prevalence of West Nile Virus (WNV) antibodies among the human population in a historical and strategic region of Turkey. A serologic survey was conducted based on suspected cases in April, 2009, in the Mesopotamia region of Turkey, in the villages that were located alongside the Zergan River. All the sera were tested by enzyme-linked immunosorbent assay ELISA (Euroimmune), and the positive samples were tested by immunofluorescent assay (IFA; Euroimmune). As confirmation, neutralizing antibodies against WNV were tested by microneutralization assay (MNTA). In total, 307 individuals were included. The MNTA test was found to be positive among 52 individuals out of 307 (17%). In multivariate analysis, age >50 [odds ratio (OR)=5.2, confidence interval (CI) 2.76-9.97, p<0.001) and being in an occupational risk group (OR=2.02, CI 1.02-4.04, p=0.044) were found to be the risk factors for WNV seropositivity with the MNTA test. The physicians in the region should be aware of the risk of WNV infection and should be alerted to detect the clinical cases.