Aim: Little is known about neonatal brain plasticity or the impact of birth mode on neurointegrity. As a reflection of neuroaxonal damage, the neuronal structural protein neurofilament light chain (NfL) has emerged as a highly specific biomarker. Our purpose was to test the hypothesis that vaginal delivery is associated with increased NfL in neonates. Methods: NfL concentrations were measured using single-molecule array immunoassay in umbilical cord serum from healthy term neonates enrolled in the prospective KUNO-Kids Health Study. NfL values were investigated for independent influencing factors using linear and logistic models, followed by post hoc propensity score-matching. Results: Of 665 neonates, n = 470 (70.7%) were delivered vaginally and n = 195 (29.3%) by cesarean section. Median serum NfL was significantly higher after vaginal delivery 14.4 pg/mL (11.6-18.5) compared to primary 7.5 pg/mL (6.1-8.9) and secondary cesarean delivery 9.3 pg/mL (7.5-12.0). Multivariable logistic regression models showed delivery mode and gestational age to be independently associated with NfL. Propensity score-matching analysis confirmed that assisted vaginal delivery generated higher NfL compared to vaginal (non-assisted), while lowest levels were associated with cesarean section. Interpretation: Our data confirm the significant impact of birth mode on neonatal NfL levels. The persistence of these differences and their potential long-term impact have yet to be investigated.