ObjectiveTo determine the effect of prophylactic dextrose gel on the infant gut microbiome.DesignObservational cohort study nested in a randomised trial.SettingThree maternity hospitals in New Zealand.PatientsInfants at risk of neonatal hypoglycaemia whose parents consented to participation in the hypoglycaemia Prevention in newborns with Oral Dextrose trial (hPOD). Infants were randomised to receive prophylactic dextrose gel or placebo gel, or were not randomised and received no gel (controls). Stool samples were collected on days 1, 7 and 28.Main outcome measuresThe primary outcome was microbiome beta-diversity at 4 weeks. Secondary outcomes were beta-diversity, alpha-diversity, bacterial DNA concentration, microbial community stability and relative abundance of individual bacterial taxa at each time point.ResultsWe analysed 434 stool samples from 165 infants using 16S rRNA gene amplicon sequencing. There were no differences between groups in beta-diversity at 4 weeks (p=0.49). There were also no differences between groups in any other microbiome measures including beta-diversity (p=0.53 at day 7), alpha-diversity (p=0.46 for day 7 and week 4), bacterial DNA concentration (p=0.91), microbial community stability (p=0.52) and microbial relative abundance at genus level. There was no evidence that exposure to any dextrose gel (prophylaxis or treatment) had any effect on the microbiome. Mode of birth, type of milk fed, hospital of birth and ethnicity were all associated with differences in the neonatal microbiome.ConclusionsClinicians and consumers can be reassured that dextrose gel used for prophylaxis or treatment of neonatal hypoglycaemia does not alter the neonatal gut microbiome.Trial registration number12614001263684.