Although there are ongoing blood donation campaigns in Nigeria, the prevalence of voluntary blood donation is about 10% and there is limited information about the determinants of blood donation behavior, especially across rural-urban geographic areas. This study examines the rural-urban differences in willingness to donate blood. A cross-sectional study addressing adults from three rural and three urban communities was performed in 2021 to evaluate willingness, knowledge, attitude and practice of blood donation. A total of 287 individuals were surveyed. Most of the respondents across all communities have never donated blood (72%). Females aged 18-25, highly educated, and from urban communities were more incline to donate blood compared to their counterparts. The main reasons for not donating blood for rural dwellers were: never thought of it (39% vs 34.7%) and no one asked (34.4% vs 17%); fear of needles was declared mostly by urban dwellers (21.8% vs 12.5%) (p=0.02). Willingness to donate blood varies across rural and urban communities and is influenced by socio-demographic characteristics. The gap between willingness to donate and actual blood donation has consequences for the establishment of blood transfusion services. Targeted public health interventions are required to enhance awareness and knowledge and modify attitudes towards blood donation.