Background Persistent pain is a leading cause of disability worldwide yet implementation of clinical guidelines that recommend a biopsychosocial approach remains a challenge in clinical practise. Limited pain understanding amongst clinicians may be partly responsible for this. Purpose of the study 1) Qualitatively explore the experience of receiving PSE, understanding of PSE and operationalisation of PSE-related principles in routine clinical practice. 2) Quantitatively explore pain knowledge, attitudes, and behaviours of general practitioners (GPs) and nurse practitioners (NPs) before and after pain science education (PSE). Methods An exploratory, single-site, mixed-methods study in north-east England. Fifteen NPs/GPs completed questionnaires and a case-vignette before and after a 70-min face-to-face PSE lecture. Qualitative data were thematically analysed from two focus groups after the intervention. Results Clinicians’ relatively high prior levels of knowledge, attitudes, and behaviour were similar after PSE. Qualitative themes described facilitation of self-reflection on pain management behaviours, and difficulties in operationalising PSE principles in practise including: limited patient rapport; short appointment times; patients’ passive and often oppositional biomedical treatment expectations; and clinicians’ lack of readily understandable language to communicate with patients. Conclusion The findings highlight the value of PSE perceived by these clinicians who were already favourably inclined towards biopsychosocial pain management. They sought more resources for their personal learning and for communication with patients. Even with such favourable disposition, the practicalities and environment of clinical practice impeded the operationalisation of PSE-related principles. Trial registration This study was prospectively registered at ClinicalTrials.Gov (NCT04587596) in October 2020.