BACKGROUND: Mosquito-borne diseases are an emerging threat in Europe. West Nile virus (WNV) is an arbovirus commonly transmitted in an enzootic cycle involving ornithophilic mosquitoes as major vectors. In recent years, global climate change has been identified as a significant driver of the increased spread of this virus. In Italy, outbreaks of WNV infection have been recorded every year in northern Italy. The Apulia region of southern Italy was not considered a high-risk region until 2023, when an unexpected increase in WNV infections occurred. The aim of this study was to evaluate the prevalence of anti-WNV antibodies in a wide sample of blood donors in the Apulia region of southern Italy enrolled between November 2023 and February 2024. In addition, the use of protective measures against mosquito bites was assessed. METHODS: A retrospective cross-sectional study was conducted on a total of 1,579 blood donors. All sera were tested for anti-WNV IgG by ELISA. Reactive serum samples were also tested by CLIA and by the plaque reduction neutralization test (PRNT). All healthy donors answered a short anonymous questionnaire. Data analysis was performed using StataMP14.0® (StataCorp LLC, CollegeStation, TX77845-4512, USA). RESULTS: The median age was 47 years (IQR: 37-53), and 75% were male. The questionnaires administered revealed that 68.6% of the subjects had not made any trips in the few weeks prior to blood sampling, and 30.5% remembered being bitten by mosquitoes in the previous 15 days or longer. While 17 samples tested by ELISA were positive for anti-WNV IgG, only six were also positive by CLIA testing. Analysis by PRNT for WNV confirmed 5 cases. The findings revealed a WNV seroprevalence of 0.32% (95% CI: 0.07-0.59). Among the subjects who tested positive, none recalled being bitten by mosquitoes or regularly using mosquito repellents. CONCLUSIONS: Our study suggests the circulation of WNV in Apulia and highlights the potential human health concerns associated with this emerging virus. Strengthening the integrated surveillance system and planning adequate preventive strategies are crucial next steps to address the potential massive spread of WNV in southern Italy.