To establish the presence of West Nile virus (WNV) infection in the animal population in Serbia after the human WNV outbreak, the presence of anti-WNV IgG antibodies was examined by commercial ELISA of blood sera samples of 130 horses collected in 2012 from 6 stables and 1 settlement in Vojvodina Province, northern Serbia. During the blood sampling, hibernating mosquitoes in the vicinity of the sampled horses were collected (31 pools from 4 locations) and tested for WNV presence by real-time RT-PCR. The presence of anti-WNV antibodies was observed in 49.23% (64/130) horses. Per stable, the percent of seropositive animals ranged from 35% to 64%. All 31 analyzed pools of hibernating mosquitoes tested negative for WNV RNA. The WNV-antibody prevalence of 49.23% obtained in horses during 2012 was much higher than the prevalence (12%) found in horses during 2009/2010. These results, including the confirmed seroconversion in eight horses that tested negative in 2010, indicated an intensive WNV circulation during 2012 in Serbia, and the necessity of implementing surveillance programs.