ABSTRACT Objectives: In experimental models, the prenatal diet influences gut microbiota composition in mothers and offspring; however, it is unclear whether this occurs in humans. We investigated the effects of a gut-focused perinatal dietary intervention on maternal and infant gut microbiota composition four weeks after birth. Methods: This randomised controlled trial randomised pregnant women to receive dietary advice as part of standard care, or additionally receive a dietary intervention focused on the Australian Dietary Guidelines and increasing prebiotic and probiotic/fermented food intakes (ACTRN12616000936426). Study assessments occurred from gestation week 26 (baseline) to four weeks postpartum (follow-up). Faecal samples, collected at baseline for mothers, and follow-up for mothers and infants, underwent 16SrRNA sequencing. The primary outcome was a between-group mean difference in infant faecal Shannon index. Secondary outcomes included between-group differences in other microbiota measures, including maternal change from baseline CLR-transformed Prevotella abundance. Results: Forty-four women and 45 infants completed the study. The mean Shannon index of infants in the intervention group was −0.35 (95% CI: −0.64, −0.06, SD: 0.52) units lower than control group infants, corresponding to a medium effect size (Cohen’s D: −0.74, 95% CI: −1.34, −0.13). The findings were similar using other metrics of α-diversity. There were no between-group differences in β-diversity, nor any differentially abundant taxa in infants. The intervention increased abundances of the genus Prevotella in mothers compared to controls. Discussion: This gut-focused perinatal dietary intervention was associated with differences in the maternal and infant gut microbiota composition. Larger studies are required to replicate and extend these findings.