BACKGROUND: Health care practitioners' knowledge and attitudes influence patients' beliefs and health outcomes in musculoskeletal (MSK) pain. It is unclear to what extent physiotherapists undertaking a postgraduate master in manual therapy (MT students) possess the knowledge and attitudes toward pain neuroscience to be able to apply the biopsychosocial model in patients with MSK pain. OBJECTIVES: The aim of this study was to assess the knowledge and attitudes toward pain neuroscience in MT students. DESIGN: A cross-sectional study. METHOD: Self-reported knowledge and attitudes were measured among students (n = 662) at baseline and in all years of the MT postgraduate programs in the Netherlands. The Knowledge and Attitudes of Pain questionnaire (KNAP) was used as a primary measure. Difference in KNAP-scores between baseline (0), year 1, year 2 and year 3 was tested using a one-way ANOVA (hypothesis: 0 < 1<2 < 3). A two factor ANOVA was used to determine the interaction effect of focused pain education and year in the curriculum with KNAP. RESULTS: There was an overall significant difference of KNAP scores with a medium effect size (F(3, 218.18) = 13.56, p < .001, ω2 = 0.059). Differences between years ranged from small to medium. Interaction effect of knowledge and attitudes and focused pain education was significant with a small effect size (F(6) = 2.597, p = .017, ω2 = 0.012). Sensitivity analyses were consistent with the main results. CONCLUSIONS: Positive differences in knowledge and attitudes toward pain neuroscience in MT students occur between the progressing years of the curriculum. Differences may be related to the provision of focused pain education.