Background: A disturbance in the early colonisation of the gut by microorganisms is associated with an aberrant innate immune system and a variety of clinical conditions later in life. Several factors are considered to influence this initial colonisation, including maternally administered antibiotics during pregnancy and delivery. Recent revisions to international obstetric guidelines have resulted in the exposure of all infants born by caesarean section (CS) to broad-spectrum antibiotics perinatally. To date, the consequences of these new guidelines on neonatal gut colonisation and the associated short- and long-term health implications have not yet been addressed. The aim of this study is to investigate the influence of the timing of antibiotic administration during CS to the mother on the course of neonatal intestinal colonisation up to 2 years of age. Methods/design: This single-centre randomised controlled trial will recruit 40 women scheduled for an elective CS. The subjects will be randomised to receive 1500 mg of cefuroxime intravenously either prior to the skin incision (n = 20) or after clamping of the umbilical cord (n = 20). Levels of cefuroxime in cord blood will be determined for exposed neonates. Faecal samples from the children will be collected on days 1, 7 and 28 days and at 2 years old and analysed by 16S sequencing. Shannon-diversity indices, absolute and relative abundances, and unsupervised and supervised classification methods will be used to evaluate the effect of the timing of intrapartum cefuroxime administration on the composition of the microbiota. The outcomes for both study groups will be compared to the intestinal microbiota of vaginally born infants (n = 20). To detect possible effects on health state, a questionnaire on health-related issues will be taken at the age of 2 years. Discussion: In the proposed study, changes in the intestinal microbiota of 40 children born by CS will be followed until the age of 2 years. Research on this topic is necessary since significant effects relating to the timing of antibiotic administration on microbial colonisation may conflict with the current guidelines, as this may have health consequences later in life. Trial registration: Netherlands Clinical Trial Registry, NTR6000. Retrospectively registered on 25 July 2016.