BACKGROUND: Mother-to-newborn transmission of obesity-associated microbiota may be modified by birth mode (vaginal vs. Cesarean delivery). Prospective data to test this hypothesis are still sparse. OBJECTIVE: To examine prospective associations of maternal pre-pregnancy BMI and gestational weight gain with the infant gut microbiome by birth-mode strata. SUBJECTS/METHODS: In 335 mother-infant pairs in the New Hampshire Birth Cohort, we ascertained data from questionnaires and medical records, and generated microbiome data from 6-week-old infants' stool using Illumina 16s rRNA gene sequencing (V4-V5 region). Analyses were stratified by birth mode and conducted before and after adjusting for potential confounders, which included maternal age, education, parity, and Mediterranean diet score. RESULTS: Among 335 mothers, 56% had normal pre-pregnancy BMI ( < 25, referent), 27% were overweight (BMI 25-30), and 18% obese (BMI > 30). Among the 312 mothers with weight gain data, 10% had inadequate weight gain, 30% adequate (referent), and 60% excess. Birth mode modified associations of pre-pregnancy BMI with several genera, including the most abundant genus, Bacteroides (P for interaction = 0.05). In the vaginal-delivery group, maternal overweight or obesity was associated with higher infant gut microbiome diversity and higher relative abundance of 15 operational taxonomic units (OTUs), including overrepresentation of Bacteroides fragilis, Escherichia coli, Veillonella dispar, and OTUs in the genera Staphylococcus and Enterococcus. In the Cesarean-delivered group, there were no significant associations of pre-pregnancy BMI with infant microbiome (alpha) diversity or OTUs. Gestational weight gain was not associated with differential relative abundance of infant gut microbial OTUs or with measures of microbial diversity in infants delivered vaginally or by Cesarean section. CONCLUSIONS: Among vaginally-delivered infants, maternal overweight and obesity was associated with altered infant gut microbiome composition and higher diversity. These associations were not observed in Cesarean-delivered infants, whose microbiome development differs from vaginally-delivered infants. Our study provides additional evidence of birth-mode dependent associations of maternal body weight status with the infant gut microbiota. The role of these associations in mediating the intergenerational cycle of obesity warrants further examination.