Background: Maternal obesity is associated with adverse pregnancy outcomes. Probiotic supplementation during pregnancy may have positive effects on blood glucose, gestational weight gain (GWG), and the risk of gestational diabetes mellitus (GDM). The primary aim was to determine the feasibility of probiotic intervention in obese pregnant women from the early second trimester until delivery. The secondary aim was to investigate the effect of daily probiotic supplementation on GWG, maternal glucose homeostasis, infant birthweight, and maternal gut microbiota. We carried out a randomized double-blinded placebo-controlled study in 50 obese pregnant women. Participants were randomly allocated to two treatment groups, multi-strain probiotic [Vivomixx®] or placebo at 14–20 weeks of gestation until delivery. Participants were followed with two pre-delivery visits at gestational week 27-30 and 36-37 and with one post-delivery visit 2-3 days after birth. All visits included blood and fecal sampling. An oral glucose tolerance test was performed at inclusion and gestational week 27-30. Results: Forty-nine participants completed the study. Thirty-eight participants took more than 80% of the Vivomixx® capsules (n=21), placebo (n=17). There was no significant difference in HbA1c levels and the occurrence of GDM between groups. There was no significant difference in GWG and infant birth weight between groups in intention to treat analysis. There was, however, a lower mean GWG (11.9 vs 13.0 kg) and lower mean infant birthweight (3554 vs 3658 g) in the probiotic group in the per protocol analysis, due to sample size this difference did not reach statistical significance. Fecal microbiota analyses showed an overall increase in α-diversity over time in the Vivomixx® group only (p=0.016). Conclusions: Administration of probiotics during pregnancy is feasible in obese women. Multi-strain probiotic can modulate the gut microbiota in obese women during pregnancy. A larger study population is needed to uncover whether the results regarding lower GWG and infant birth weight after probiotic supplementation are significant. Trial registration: ClincalTrials.gov Identifier: NCT02508844, registered on May 11, 2015.