Training for knowledge and attitude change can be a key component of improving animal care and welfare. Much of the available research is focused upon the livestock industry but can be applied to other animal industries. In the research and teaching industry (RTI), training is an important legal, regulatory, professional, and ethical responsibility as well as a forming part of the social licence for industry. A comparative study was developed to explore the impact of training targeting attitudes and knowledge on participants’ use of grimace scales (GS) in the Australian and New Zealand (ANZ) RTI. Participants were recruited into training sessions that excluded (control) or included (enhanced) training targeting attitudes. Prior to any training, cohorts received a mixed-methods, quantitative-qualitative questionnaire to determine baseline knowledge and attitudes towards GS, animal welfare, and pain management. Post-training, participants completed questionnaires to investigate changes in their knowledge or attitudes. Training (control and enhanced) resulted in up to eight times more positive attitudes and knowledge towards the GS and pain management. Only enhanced training for attitudes offered additional key benefits with participants being up to three times more confident in GS and more likely to offer pain relief. The results from this study demonstrate the ability for GS training to improve human-animal interactions with potential improvements in pain management, animal welfare, the 3Rs, and research. These outcomes support the greater body of literature and best practice recommendations for RTIs to require and implement training programmes that incorporate attitudinal training in animal care and use programmes.