ObjectiveThis meta-analysis was conducted to evaluate the overall effect of direct-fed microbial (DFM) or probiotic supplementation on the log concentrations of culturable gut microbiota in broiler chickens.MethodsRelevant studies were collected from PubMed, SCOPUS, Poultry Science Journal, and Google Scholar. The studies included controlled trials using DFM supplementation in broiler chickens and reporting log concentrations of the culturable gut microbiota. The overall effect of DFM supplementation was determined using standardized mean difference (SMD) with a random-effects model. Subgroups were analyzed to identify pre-specified characteristics possibly associated with the heterogeneity of the results. Risk of bias and publication bias were assessed.ResultsEighteen taxa of the culturable gut microbiota were identified from 42 studies. The overall effect of DFM supplementation on the log concentrations of all 18 taxa did not differ significantly from the controls (SMD = −0.06, 95% confidence interval [−0.16, 0.04], p = 0.228, I2 = 85%, n = 699 comparisons), but the 18 taxa could be further classified into three categories by the direction of the effect size: taxa whose log concentrations did not differ significantly from the controls (category 1), taxa whose log concentrations increased significantly with DFM supplementation (category 2), and taxa whose log concentrations decreased significantly with DFM supplementation (category 3). Category 1 comprised nine taxa, including total bacterial counts. Category 2 comprised four taxa: Bacillus, Bifidobacterium, Clostridium butyricum, and Lactobacillus. Category 3 comprised five taxa: Clostridium perfringens, coliforms, Escherichia coli, Enterococcus, and Salmonella. Some characteristics identified by the subgroup analysis were associated with result heterogeneity. Most studies, however, were present with unclear risk of bias. Publication bias was also identified.ConclusionDFM supplementation increased the concentrations of some beneficial bacteria (e.g. Bifidobacterium and Lactobacillus) and decreased those of some detrimental bacteria (e.g. Clostridium perfringens and Salmonella) in the guts of broiler chickens.