An anthroposophic lifestyle, which has been associated with reduced allergy risk in children, has several characteristics that could influence gut microbiota. This study aimed to investigate the impact of anthroposophic lifestyle as well as specific early life exposures, on the gut microbiota. In total, 665 stool samples from 128 mother-child pairs from the ALADDIN birth cohort study were included. Samples collected from infants at ages six days, three weeks, two months and six months and from their mothers before and after delivery, respectively, were analyzed using 454-pyrosequencing. Information regarding lifestyle exposures was collected prospectively through interviews and questionnaires. Six month-old infants in anthroposophic families had a significantly higher abundance of Bifidobacterium and lower abundances of Bacteroides and Veillonella. Caesarean section and breastfeeding had a significant impact on the microbiota whereby caesarean section was primarily associated with delayed colonization of Bifidobacterium and Bacteroides, whereas breastfed children had a higher relative abundance of Bifidobacterium and a lower abundance of Clostridiales. However, despite large differences in lifestyle exposures, we determined no significant differences in the gut microbiota between the anthroposophic and non-anthroposophic mothers or their infants’ before six months of age.This article is protected by copyright. All rights reserved.