We use self-determination theory to extend the conceptual understanding of flu vaccine hesitancy among health professionals. The scale sheds light on the role played by motivational factors above and beyond traditional cognitive factors such as biased risk judgements and health beliefs.Across five phases using data from 718 healthcare professionals we establish factor structure, reliability, discriminant, convergent, criterion-related, incremental validity, and measurement invariance of the Treatment Self-Regulation Questionnaire assessing healthcare professionals’ motivation for flu vaccination scale (TSRQ-Flu). In addition to the four factors of the TSRQ-Flu (autonomous, introjection, external and amotivation regulations), we assess intentions to vaccinate, past vaccination behaviour and validate the scale using measures of cognitive empowerment, vaccine attitudes and social desirability. Our findings indicate that the newly developed 11-item scale is distinct from and contributes over and beyond other psychosocial measures of flu vaccination intentions and can be used to understand the motivation of both vaccinated and not-vaccinated healthcare professionals. This new scale has the potential to make a marked change in the conceptualisation of the roots of vaccine hesitancy among healthcare professionals and aid healthcare managers in developing evidence-based interventions to promote vaccination among their staff.