Animal welfare awareness (AWA) during transportation and in markets is a critical concern in livestock production, influencing the health of animals and other outcomes for stakeholders. Nevertheless, it remains understudied in many developing regions. This study investigates the level of awareness and practices regarding animal welfare during and after transportation among primary stakeholders — sellers, drivers, and buyers — in three livestock markets in Nigeria: Achida, Ikorodu Sabo, and Amansea. A structured survey focusing on the stakeholders’ familiarity with the concept of animal welfare, the Five Freedoms, the Animal Diseases (Control) Act, and encounters with veterinary control posts was conducted across the selected markets between February and July 2024. Furthermore, stakeholders were also questioned about barriers to improving practices. Descriptive and inferential statistical analyses were performed to explore associations between the dichotomised awareness of animal welfare and key variables. A significant association between AWA and market location was revealed, but not with occupation. Further analysis showed that dichotomised awareness of Veterinary Control Posts (VCPs) and the Animal Diseases (Control) Act had significant negative associations with AWA, suggesting complex relationships between legal knowledge and familiarity with the concept of animal welfare. Additionally, transport-related mortality was reported by 70.7% of respondents, with overcrowding and sickness identified as primary causes. However, significant barriers, including economic constraints and a lack of authority to mandate standards, were the leading challenges often faced by stakeholders. The findings underscore the need for targeted interventions and policy reforms, including increased enforcement.