Introduction: Bifidobacterium breve M-16V has been used as a probiotic in preterm infants. Probiotic strain-specific data are essential to guide clinical practice. Objective: To assess effects of B breve M-16V in preterm neonates. Design: A systematic review of randomized controlled trials (RCTs) and non-RCTs of B breve M-16V in preterm infants was conducted. Multiple databases, proceedings of Pediatric Academy Society, and other relevant conferences were searched in September 2016 and on January 5, 2017. Results: Five RCTs (n = 482) and 4 non-RCTs (n = 2496) were included. Of the 5 RCTs, 4 carried high/unclear risk of bias in many domains. Meta-analysis (fixed effects model) of RCTs showed no significant benefits on stage ≥2 necrotizing enterocolitis, late-onset sepsis, mortality, and postnatal age at full feeds. Meta-analysis of non-RCTs showed significant benefits on (1) late-onset sepsis—3 studies (n = 2452), odds ratio = 0.56 (95% CI, 0.45–0.71), P <.0001; (2) mortality—2 studies (n = 2319), odds ratio = 0.61 (95% CI, 0.44–0.84), P =.002; and (3) postnatal age at full feeds (days)—2 studies (n = 361), mean difference, −2.42 (95% CI, −2.55 to −2.3), P <.00001. There were no adverse effects from B breve M-16V. On Grading of Recommendations, Assessment, Development, and Evaluation analysis, the overall quality of evidence was deemed very low. Conclusions: Current evidence is limited regarding the potential of B breve M-16V in preterm neonates. Adequately powered, preferably cluster RCTs are needed to confirm these findings.