Mosquito community composition plays a central role in the transmission of zoonotic vector-borne pathogens. We evaluated how the mosquito community affects the seroprevalence of West Nile virus (WNV) in house sparrows along an urbanisation gradient in an area with the endemic circulation of this virus. We sampled 2544 birds and 340829 mosquitoes in 45 localities, analysed in 15 groups, each containing one urban, one rural and one natural area. WNV seroprevalence was evaluated using an epitope-blocking ELISA kit and a micro virus-neutralization test (VNT). The presence of WNV antibodies was confirmed in 1.96% and 0.67% of birds by ELISA and VNT, respectively. The VNT-seropositive birds were captured in rural and natural areas, but not in urban areas. Human population density was zero in all the localities where VNT-positive birds were captured, which potentially explains the low incidence of human WNV cases in the area. The prevalence of neutralizing antibodies against WNV was positively correlated with the abundance of the ornithophilic Culex perexiguus but negatively associated with the abundance of the mammophilic Ochlerotatus caspius and Anopheles atroparvus. These results suggest that the enzootic circulation of WNV in Spain occurs in areas with larger populations of Cx. perexiguus and low human population densities.