Background: The burden of Road Traffic Accidents (RTA) has been on the increase in Nigeria. First-Aid (FA) service is one of the key measures for reducing RTA-related injuries and complications. Information about factors, which could influence FA response by residents of communities along major highways in Nigeria, is insufficient. Objective: This study aims to examine factors influencing FA assistance to accident victims and action taken by residents of selected communities along Lagos-Ibadan expressway. Methodology: A cross-sectional survey conducted in six randomly selected communities along Ogun state axis of the Lagos-Ibadan expressway. A three-stage sampling technique was used to select 599 consenting respondents from the communities based on proportionate population. An interviewer- administer- questionnaire containing questions on awareness of FA, actions taken when accidents occurred, A 16-point FA knowledge scale was used to collect the data. Knowledge scores of ≤ 10 and ≥11were rated as poor and good respectively. Quantitative data were analyzed using descriptive statistics, Chi-square, and ANOVA. The level of significance was set at 0.05. Result: Majority (79.1%) of the respondents had ever witnessed an accident along the expressway and 46.7% had ever assisted accident victims. Overall mean knowledge score was 6.0 ± 2.0 with 95.2% of respondents showing poor knowledge of FA. Factors which cause delay in conveying accidents victim to the hospital included: fear of negative reactions from the police (24.0%); traffic congestion (18.5%) and lack of vehicle (16.2%). Conclusion: There was poor knowledge of First Aid. Logistics problems are the main barriers to the provision of First Aid services to accident victims on the expressway. Sensitization and training related to First Aid should be instituted at the community level.