Background and Objective: 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. The aim of the study is to assess the rural-urban differences in willingness, knowledge, attitude and practice of blood donation. Methods: A cross-sectional study addressing adults from three rural and three urban communities in Ibadan region was performed in 2021 using a structured interviewer-administered questionnaire. The study subjects were selected through a multi-stage sampling method. Results: A total of 287 individuals were surveyed, of which 72% have never donated blood. The majority knew about blood donation (83.6%), considered it a safe practice (88.8%), and knew where to donate blood, especially rural dwellers (78.2% vs. 57.5%; p=0.001). However, urban dwellers were more willing to donate blood (67% vs. 58.6%; p=0.03). 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%); most urban dwellers were afraid of needles (21.8% vs. 12.5%) (p=0.02). Of 76 blood donors, 68.4% had donated once and 47.4% donated for relatives or friends. Most rural dwellers declared that blood donors should be compensated (82.8% vs. 48.5%; p<0.001). Conclusions: Willingness to donate blood varies across rural and urban communities and is influenced by several factors, including cultural context, the need for motivations and incentives. Consequently, willingness to donate blood does not translate to active practice of blood donation, with consequences for the establishment of blood transfusion services. Public health interventions are required to enhance awareness and knowledge about blood donation and should be targeted to the cultural context and geographical area. The provision of incentives could be taking into consideration in areas with high blood shortage.