IMPORTANCE: The intestinal microbiome plays a critical role in infant development, and delivery mode and feeding method (breast milk vs formula) are determinants of its composition. However, the importance of delivery mode beyond the first days of life is unknown, and studies of associations between infant feeding and microbiome composition have been generally limited to comparisons between exclusively breastfed and formula-fed infants, with little consideration given to combination feeding of both breast milk and formula. OBJECTIVE: To examine the associations of delivery mode and feeding method with infant intestinal microbiome composition at approximately 6 weeks of life. DESIGN, SETTING, AND PARTICIPANTS: Prospective observational study of 102 infants followed up as part of a US pregnancy cohort study. EXPOSURES: Delivery mode was abstracted from delivery medical records, and feeding method prior to the time of stool collection was ascertained through detailed questionnaires. MAIN OUTCOMES AND MEASURES: Stool microbiome composition was characterized using next-generation sequencing of the 16S rRNA gene. RESULTS: There were 102 infants (mean gestational age, 39.7 weeks; range, 37.1-41.9 weeks) included in this study, of whom 70 were delivered vaginally and 32 by cesarean delivery. In the first 6 weeks of life, 70 were exclusively breastfed, 26 received combination feeding, and 6 were exclusively formula fed. We identified independent associations between microbial community composition and both delivery mode (P.001; Q .001) and feeding method (P = .01; Q .001). Differences in microbial community composition between vaginally delivered infants and infants delivered by cesarean birth were equivalent to or significantly larger than those between feeding groups (P =?.003). Bacterial communities associated with combination feeding were more similar to those associated with exclusive formula feeding than exclusive breastfeeding (P =?.002). We identified 6 individual bacterial genera that were differentially abundant between delivery mode and feeding groups. CONCLUSIONS AND RELEVANCE: The infant intestinal microbiome at approximately 6 weeks of age is significantly associated with both delivery mode and feeding method, and the supplementation of breast milk feeding with formula is associated with a microbiome composition that resembles that of infants who are exclusively formula fed. These results may inform feeding choices and shed light on the mechanisms behind the lifelong health consequences of delivery and infant feeding modalities.