Abstract Background Despite extensive investigation of ergonomic risk factors for spinal pain in healthcare workers, limited knowledge of psychological risk factors exists. Aims To assess the prospective association of mental health and vitality with development of spinal pain in healthcare workers. Methods A prospective cohort study was carried out involving 1950 healthcare workers from 19 hospitals in Denmark. Assessments were done at baseline and at 1-year follow-up. Mental health and vitality were measured using the Short Form-36 Health Survey, while spinal pain intensity was measured using a 0–10 scale in the low-back, upper-back and neck, respectively. Cumulative logistic regressions adjusted for several confounding factors were applied, reporting risk estimates as odds ratios (ORs) and 95% confidence intervals (CIs). Results Using good mental health as reference, moderate (but not poor) mental health at baseline was associated with increased pain intensity in the low-back (OR: 1.41 [95% CI: 1.21–1.77]), upper-back (OR: 1.63 [95% CI: 1.31–2.02]) and neck (OR: 1.31 [95% CI: 1.07–1.61]) at 1-year follow-up. Likewise, using high vitality as reference, both moderate and low vitality at baseline were associated with increased pain intensity in the low-back (OR: 1.54 [95% CI: 1.22–1.94] and OR: 2.34 [95% CI: 1.75–3.12], respectively), upper-back (OR: 1.72 [95% CI: 1.34–2.23] and OR: 2.46 [95% CI: 1.86–3.25], respectively) and neck (OR: 1.66 [95% CI: 1.34–2.06] and OR: 2.06 [95% CI: 1.61–2.63], respectively) at 1-year follow-up. Conclusions Compared to healthcare workers with good mental health and high vitality, those with moderate mental health and low/moderate vitality, respectively, were more likely to increase spinal pain intensity at 1-year follow-up. These components should also be considered in the prevention of spinal pain in healthcare workers.