This systematic review and meta-analysis aimed to determine if probiotic supplementation in pregnancy reduced maternal Group B streptococcus (GBS) recto-vaginal colonization in pregnant women at 35–37 weeks of gestation. Electronic databases (i.e., PubMed, MEDLINE, ClinicalTrials.gov, ScienceDirect, and the Cochrane Library) were searched from inception up to February 2022. We included RCTs assessing the effects of probiotic supplementation in pregnancy on GBS recto-vaginal colonization. The primary outcome was GBS-positive recto-vaginal cultures performed at 35–37 weeks of gestation. Secondarily, we evaluated obstetric and short-term neonatal outcomes. A total of 132 publications were identified; 9 full-length articles were reviewed to finally include 5 studies. Probiotic supplementation reduced vaginal GBS colonization: the GBS positive culture rate was estimated at 31.9% (96/301) in the intervention group compared to 38.6% (109/282) in the control group (OR = 0.62, 95% CI 0.40–0.94, I2 4.8%, p = 0.38). The treatment started after 30 weeks of gestation and was more effective in reducing GBS colonization (OR 0.41, 95% CI 0.21–0.78, I2 0%, p = 0.55). Probiotic administration during pregnancy, namely in the third trimester, was associated with a reduced GBS recto-vaginal colonization at 35–37 weeks and a safe perinatal profile. Whether this new strategy could reduce the exposition of pregnant women to significant doses of antibiotics in labor needs to be evaluated in other trials.