Background <p>Pain neuroscience education (PNE) promotes a biopsychosocial understanding of pain among health-professional students, yet evidence for its long-term effects outside high-income countries remains limited. This study evaluated the immediate and six-month effects of a single PNE lecture on pain knowledge, attitudes, beliefs, and clinical recommendations in Jordanian physiotherapy students.</p> Methods <p>A parallel-group randomized controlled trial was conducted with first- and second-year physiotherapy students. Students were randomized (1:1; 60 per group) to a 70-minute PNE lecture or an attention-matched musculoskeletal red flags lecture. Primary outcomes were pain knowledge (Revised Neurophysiology of Pain Questionnaire [RNPQ]) and pain-related attitudes (modified Health Care Providers’ Pain and Impairment Relationship Scale [HC-PAIRS]). The secondary outcome was guideline-concordant recommendations assessed using a validated vignette. Analyses included linear and logistic mixed-effects models, with responder analyses using minimal clinically important difference thresholds to estimate numbers needed to treat (NNTs).</p> Results <p>All students (<i>N</i> = 120) completed the study with no attrition. Significant group × time interactions were observed for pain knowledge and attitudes (both <i>p</i> &lt; 0.001). Post-intervention, the PNE group demonstrated higher pain knowledge scores than the control group (adjusted mean difference = 2.46, 95% CI 1.68–3.24) and more favorable attitudes (adjusted mean difference = − 7.54, 95% CI − 10.62 to − 4.46). At six months, knowledge gains remained statistically significant (1.20, 95% CI 0.25–2.15; <i>p</i> = 0.020), while the between-group difference for attitudes was no longer statistically significant (–3.60, 95% CI − 7.25 to 0.05; <i>p</i> = 0.060). Guideline-concordant clinical recommendations improved immediately after PNE (adjusted ORs 2.95–6.18) but were not sustained at follow-up (all <i>p</i> &gt; 0.05). Responder analyses demonstrated substantial immediate educational benefit (NNT 2.7 for knowledge, 3.6 for attitudes), which decreased at six months, consistent with attenuation of intervention effects over time.</p> Conclusion <p>A single 70-minute PNE session produced significant and clinically meaningful immediate improvements in pain knowledge, attitudes, and guideline-concordant clinical recommendations. At six months, pain knowledge remained significantly improved, whereas the remaining effects attenuated. These findings support consideration of embedding PNE early within pre-licensure physiotherapy curricula. Future research should determine whether recursive reinforcement improves longer-term retention of educational gains.</p> Trial registration <p>The study protocol was prospectively registered at ClinicalTrials.gov (NCT06821022) on February 6, 2025.</p>