Improvements in growth performance and health are key goals in broiler chicken production. Inclusion of prebiotic galacto-oligosaccharides in broiler feed enhanced the growth rate and feed conversion of chickens relative to a calorie-matched control diet. Comparison of the cecal microbiota identified key differences in abundance of Lactobacillus spp. Increased levels of L. johnsonii in GOS-fed juvenile birds at the expense of L. crispatus was linked to improved performance (growth rate and market weight). Investigation of the innate immune responses highlighted increases of ileal and cecal IL-17A gene expression counterposed to a decrease in IL-10 and IL-17F. Quantification of the autochthonous Lactobacillus ssp. revealed a correlation between bird performance and L. johnsonii abundance. Shifts in the cecal populations of key Lactobacillus spp. of juvenile birds primed intestinal innate immunity without harmful pathogen challenge.