Objective: 1) To investigate the effects of a brief pain neuroscience education (PNE) lecture on multi-disciplinary healthcare students’ knowledge, attitudes and behaviours towards people with pain post intervention and at 6-months follow-up, 2) To explore students’ perceptions of PNE. Design: Mixed-methods randomized controlled trial Setting: UK university. Participants: Thirty-seven students (30♀, mean age 30years) from six healthcare disciplines. Intervention: 70-minute PNE lecture (intervention group) or a 70-minute control education. Main outcome measures: 1) Knowledge: The Revised Pain Neurophysiology Quiz (RPNQ); 2) Attitudes: Health Care Providers’ Pain and Impairment Relationship Scale (HC-PAIRS); 3) behaviours: A case vignette to assess clinical recommendations; and 4) thematic analysis of semi-structured interviews (n=12). Results: The intervention group increased knowledge compared to the control, post-intervention [mean difference 3.7 (95% CI, 2.4, 5.0), P<0.001] but not at 6-months (0.1 (-1.1 , 1.3), P=0.860). Greater improvements in attitudes for the intervention group were seen post-intervention [-10.4 (-16.3 , -4.6), P<0.001] and at 6-months [-5.8, (-11.5 , -0.2), P<0.044]. There was no difference in behaviours between groups. Thematic analysis identified increased patient empathy, partial and patchy reconceptualisation of pain and increased confidence in recommending an active management programme following PNE. Conclusion: This study adds to existing knowledge by demonstrating that a 70-minute PNE lecture can have a short-term effect on knowledge and positively shift attitudes towards people with pain in the short and medium-term. It also resulted in some students’ reconceptualisation of pain, increased empathy, and confidence to recommend activity. The effect of PNE on clinical behaviours was unclear.