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Gestational diabetes mellitus (GDM) is a worldwide public health problem affecting up to 27% of pregnancies with high predictive values for childhood obesity and inflammatory diseases. Compromised seeding of the infant gut microbiota is a risk factor for immunologic and metabolic diseases in the offspring; however, how GDM along with maternal obesity interact to alter colonization remains unknown. We hypothesized that GDM individually and in combination with maternal overweight/obesity would alter gut microbial composition, diversity, and short-chain fatty acid (SCFA) levels in neonates. We investigated 46 full-term neonates born to normal-weight or overweight/obese mothers with and without GDM, accounting for confounders including cesarean delivery, lack of breastfeeding, and exposure to antibiotics. Gut microbiota in 2-week-old neonates born to mothers with GDM exhibited differences in abundance of 26 microbial taxa; 14 of which showed persistent differential abundance after adjusting for pre-pregnancy BMI. Key pioneering gut taxa, including potentially important taxa for establishing neonatal immunity, were reduced. Lactobacillus , Flavonifractor , Erysipelotrichaceae , and unspecified families in Gammaproteobacteria were significantly reduced in neonates from mothers with GDM. GDM was associated with an increase in microbes involved in suppressing early immune cell function ( Phascolarctobacterium ). No differences in infant stool SCFA levels by maternal phenotype were noted; however, significant correlations were found between microbial abundances and SCFA levels in neonates. Our results suggest that GDM alone and together with maternal overweight/obesity uniquely influences seeding of specific infant microbiota in patterns that set the stage for future risk of inflammatory and metabolic disease., Competing Interests: JF is a consultant to the scientific advisory board of Janssen Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Soderborg, Carpenter, Janssen, Weir, Robertson, Ir, Young, Krebs, Hernandez, Barbour, Frank, Kroehl and Friedman.)