The incidence of ‘traumatic’ lumbar puncture (LP; CSF red cells > 500/mm) has been reported to be 35–46% in the neonatal period. A traumatic LP incurs challenges in diagnosis and management of the underlying condition and increases the risk of complications. We aimed to assess the benefits of a smaller outer diameter, larger gauge 25G needle in reducing the incidence of traumatic LPs compared with the standard 22G LP needle. This prospective observational study compared data from two consecutive epochs. Epoch 1 (Control, April 2016–October 2016), 22G needle for LP as standard practice. Epoch 2 (Intervention, November 2016–October 2017) 25G needle used for LP. Primary outcome was the incidence of traumatic LP. Multiple logistic regression analyses were conducted adjusting for corrected gestational age (CGA) at LP, proceduralist experience and need for ventilation as an indicator of illness. There were 240 LPs during the study period involving 361 attempts (22G, n = 228; 25G, n = 133). Median gestation at birth (P = 0.617) and CGA at LP (P = 0.163) were comparable. Multivariate analysis revealed lower incidence of traumatic LP using 25G needle (P < 0.001). Incidence of obtaining a successful CSF sample was similar between groups (P = 0.944). Proceduralist experience (P = 0.189) and neonatal illness (P = 0.801) were not significant factors. Conclusion: Our results showed that traumatic LPs were ~ 50% less common with 25G vs 22G needles while retaining a comparable success rate. Dry taps were more likely among the 25G group.What is Known:• The incidence of neonatal ‘traumatic’ lumbar puncture (CSF red cells > 500/mm) has been reported to be 35–46%.• A traumatic lumbar puncture incurs challenges in diagnosis and management of the underlying condition and increases the risk of complications.What is New:• Multivariate analysis revealed lower incidence of traumatic lumbar puncture using 25G needle (vs 22G).• Incidence of obtaining a successful CSF sample was similar between groups.