AIM: The objective of this study was to investigate the involvement of the motor fibres of the corpus callosum after unilateral neonatal arterial ischemic stroke (NAIS) of the middle cerebral artery territory and the relationship to both ipsilesional and contralesional hand function. METHOD: Using high-resolution structural magnetic resonance imaging (MRI), functional MRI, and magnetic resonance diffusion-tractography, we compared the midsagittal area of the motor part of the corpus callosum (defined by the fibres connecting the precentral gyri) between 33 7-year-old children after unilateral NAIS and 31 typically developing 7-year-old children. Hand motor performance was assessed by the box and blocks test. RESULTS: Children after NAIS showed on average significantly smaller motor corpus callosum area compared to typically developing children (p<0.001, without differences of the non-motor corpus callosum area). In addition, there was a significant positive association between the motor part of the corpus callosum and both contralesional (Pr(>|t|)=0.034) and ipsilesional hand motor performance (Pr(>|t|)=0.006) after controlling for lesion volume and sex. In a post-hoc analysis the additional contribution of corticospinal tract damage was evaluated. INTERPRETATION: Compared to typically developing children, children after NAIS exhibited a smaller motor part of their corpus callosum associated with reduced contralesional but also ipsilesional manual dexterity. These results indicate that the affection of transcallosal motor fibres in unilateral NAIS might be of functional relevance and an important part of the involved structural network that should be elucidated in further studies. TRIAL REGISTRATION: ClinicalTrials.gov NCT02511249. © 2017 Mac Keith Press.