Aims/hypothesis <p>The breastmilk microbiome plays a crucial role in gut microbial colonisation and immune development, but little is known about how it is influenced by type 1 diabetes.</p> Methods <p>We conducted a longitudinal 16S rRNA gene sequencing study of milk from women with type 1 diabetes (<i>n</i>=69 pregnancies; 174 samples) and women who did not have type 1 diabetes (<i>n</i>=49 pregnancies; 123 samples), collected at seven timepoints from birth to 15 months postpartum. Alpha diversity (richness, inverse Simpson evenness) was analysed by generalised linear mixed models, beta diversity was analysed by Bray–Curtis dissimilarities and PERMANOVA, and differential abundance was analysed by limma. Additionally, we examined associations with maternal genetic risk score (GRS), maternal HLA type, glycaemic management (HbA_1c) and breastmilk secretory IgA (sIgA), and performed a parallel analysis for the infant stool microbiome.</p> Results <p>A significant interaction between type 1 diabetes status and timepoint was observed for alpha diversity, both richness (<i>p</i>=0.01) and inverse Simpson diversity (<i>p</i>=0.003), indicating distinct temporal trajectories between women with and without type 1 diabetes. In those without type 1 diabetes, richness increased significantly between birth and 1&#xa0;week postpartum, but this early increase was delayed in women with type 1 diabetes to between 1&#xa0;week and 3&#xa0;months postpartum (<i>p</i>=0.002). Beta diversity analysis revealed earlier and more extensive compositional shifts in women without type 1 diabetes compared to those with type 1 diabetes. These differences persisted after adjusting for Caesarean delivery, BMI, parity and infant sex, and were not attributable to a delay in initiating breastfeeding. Taxa with delayed enrichment in women with type 1 diabetes included <i>Streptococcus</i> spp. and <i>Rothia mucilaginosa</i>, which metabolise human milk oligosaccharides to short-chain fatty acids to promote development of the infant’s gut barrier and immune system. Maternal GRS, HLA, HbA_1c or sIgA were not associated with milk microbiota diversity trajectories. In infant stool samples, alpha diversity did not differ between exposure groups, and showed no evidence of delayed maturation. Beta diversity revealed an early compositional shift between birth and 1&#xa0;week postpartum only in infants born to women without type 1 diabetes. Similarly, significant taxonomic changes between birth and 1&#xa0;week postpartum were detected only in infants born to women without type 1 diabetes, but with some taxa differing between exposure groups at 1&#xa0;week.</p> Conclusions/interpretation <p>Maternal type 1 diabetes is associated with delayed early maturation of the breastmilk microbiome. Early compositional differences in microbiota restructuring were also observed in the infant gut, partially mirroring the pattern in the milk microbiome; however, sustained differences in infant gut microbiota diversity were not detected. Further investigation could determine whether these changes affect development of the infant’s gut and immune system.</p> Graphical Abstract <p></p>