Background: Early antibiotic treatment changes susceptibility to disease in rodent models for type 1 diabetes, but the impact that gestational antibiotics may have on pancreatic embryogenesis and β cell function later in life is still unknown. Methods: Our aim was to determine whether gestational antibiotics could change islet neogenesis and function in female non-obese diabetic mouse offspring. Stereology of pancreatic islets and insulin measurements were therefore applied to 2-week-old pups from mothers either exposed or not to a broad-spectrum cocktail of ampicillin (1 g/L), vancomycin (0.5 g/L) and neomycin (1 g/L) during pregnancy. The degree of insulitis, glycated haemoglobin (HbA1c), serum cytokines and endotoxin, fasting glucose and insulin, and glucose tolerance were tested in the prediabetic pups later in life. Inflammatory and glycaemic measurements were made on the breeding females, and the gut microbiota was analysed by 16S sequencing in both the mothers and pups. Results: Antibiotics depleted the maternal microbiome and perturbed the normal gut colonisation trajectory in the pups. While Akkermansia and Rikenellaceae were later enriched in the treated pups, Bacteroides failed to colonise post-treatment. Antibiotic treatment reduced blood glucose in the mothers, weight of the pups, and HbA1c and insulitis in the adult offspring and increased anti-inflammatory CD4⁺ natural killer T cells. This was preceded by more than 50% increase in total islet number and volume, and increased insulin levels in postnatal life. No changes were observed in their homeostatic model assessment of insulin resistance (HOMA-IR) and β cell function (HOMA-β), serum endotoxin or cytokine levels later in life, but the glucose tolerance response was altered in the treated offspring, which suggested an improvement in β cell exhaustion that may have left the islets less vulnerable to destruction. Conclusion: Our study highlights the importance of considering maternal microbiota-modulating factors as determinants that may mitigate or aggravate diabetes via changes in islet maturation and β cell function in perinatal life.