Antibiotic resistance is one of the greatest global threats we face today. Human overuse of antibiotics is a contributing factor and major behaviour change around antibiotic consumption is needed, but several challenges exist in communicating antibiotic resistance to the public. In 2018 the UK Government relaunched a national television advertisement as part of the ‘Keep Antibiotics Working’ campaign which aimed to raise awareness of antibiotic resistance and reduce public demand for antibiotics. This study evaluates what role the framing of antibiotic resistance in the advertisement played in increasing public understanding of antibiotic resistance and motivating behaviour change. The study is grounded in behaviour change and health communication theory from the field of Communication for Development, and health and social psychology theory, reflecting the need for multidisciplinary approaches to addressing antibiotic resistance. A textual analysis identified how the issue was framed in the advertisement and surveys and interviews were conducted with members of the target audience groups to analyse what effect the advertisement had on their understanding of, and attitude towards antibiotic resistance. The findings show that the framing of antibiotic resistance in the TV advertisement led to an increase in misunderstandings of what becomes resistant to antibiotics. The advertisement was helpful in highlighting the vulnerability of antibiotics and for creating a new social norm around being a responsible antibiotic user, however was interpreted as childish by participants. It did not communicate the severity of antibiotic resistance or specific risk of antibiotic overuse to the audience, or accurately reflect the audience’s existing knowledge of antibiotic resistance and current behaviours. As the severity of antibiotic resistance was not conveyed, the advertisement did not motivate a change in antibiotic seeking behaviours or attitude amongst the majority of participants. The findings did highlight knowledge gaps amongst study participants including the importance of completing a course of antibiotics as prescribed, and that it is the bacteria itself, not the person, that develops resistance, and hopes this research can inform the development of future campaigns.