Systematic reviews of randomized, controlled trials (RCTs) indicate lower mortality and necrotizing enterocolitis (NEC) and shorter time to full feeds after probiotic supplementation in preterm (<34 weeks' gestation) very low birth weight (VLBW; birth weight <1500 g) neonates. The objective of this study was to update our 2007 systematic review of RCTs of probiotic supplementation for preventing NEC in preterm VLBW neonates. We searched in March 2009 the Cochrane Central register; Medline, Embase, and Cinahl databases; and proceedings of the Pediatric Academic Society meetings and gastroenterology conferences. Cochrane Neonatal Review Group search strategy was followed. Selection criteria were RCTs of any enteral probiotic supplementation that started within first 10 days and continued for > or =7 days in preterm VLBW neonates and reported on stage 2 NEC or higher (Modified Bell Staging). A total of 11 (N = 2176), including 4 new (n = 783), trials were eligible for inclusion in the meta-analysis by using a fixed-effects model. The risk for NEC and death was significantly lower. Risk for sepsis did not differ significantly. No significant adverse effects were reported. Trial sequential analysis) showed 30% reduction in the incidence of NEC (alpha = .05 and .01; power: 80%). The results confirm the significant benefits of probiotic supplements in reducing death and disease in preterm neonates. The dramatic effect sizes, tight confidence intervals, extremely low P values, and overall evidence indicate that additional placebo-controlled trials are unnecessary if a suitable probiotic product is available.