Simple SummaryGum Arabic (GA) is considered a natural prebiotic because it contains soluble and indigestible fibers that can stimulate the growth and activity of commensal bacteria through fermentation. Administration of GA in broiler diets may promote early growth as well as the development and health of the intestine and immune functions. However, studies on the mechanism of GA on broiler chickens to improve growth performance and intestinal health are scarce. This study aimed to evaluate the growth performance, internal organs, immune-related gene expression, microbiota, and histological changes of broiler chickens fed with increasing levels of GA. We suggest that GA (0.25 to 0.75%) positively affects the performance, microbiota, immune response, morphology, and gut health of post-hatched chickens. Gum Arabic (GA) belongs to the Fabaceae family and contains indigestible soluble fibers (80–85%) that could be fermented by commensal bacteria to enhance performance, immune response, and intestinal integrity. This study aimed to investigate the effects of GA on performance, serum biochemical indicators, microbiota, immune-related gene expression, and histological changes in chickens. Six GA levels (0.0, 0.12, 0.25, 0.5, 0.75, 1.0%) were allocated using a total of 432 1-day-old male chickens (12 replicates with 6 chickens each). Growth performance was evaluated on days 10 and 24 of age. Blood parameters, organ pH levels, and intestinal health were determined on day 10 of age. Results showed that GA at 0.12% increased weight gain and 0.12 to 1.0% decreased feed intake but was best in feed conversion ratio and production efficiency except for 1.0% on day 1–10 of age. There was an increase in the thymus weight at GA level 0.25 to 0.75%. GA decreased the pH value of the proventriculus (at 0.50 and 1.0%) as well as the duodenum and cecum (at 0.12 and 1.0%). Chickens fed GA between 0.25 to 1.0% had higher protein and HDL, but lower cholesterol, LDL, and creatinine. Globulin was increased at 0.50% GA, while glucose and triglycerides were decreased (at 0.25 and 0.75% GA, respectively). The immune-related gene expression was reduced, except for 0.25% GA, which increased IL-10. Furthermore, chickens fed GA (0.25 to 0.75%) had higher Lactobacillus spp. and lower Salmonella spp. and Escherichia coli. When chickens received GA, the villus length and length to crypt ratio were higher, which also improved the integrity of intestinal epithelial cells and early duodenal development. We conclude that using GA (0.25 to 0.75%) as a natural prebiotic positively affects the performance, microbiota, immune response, morphology, and gut health of post-hatched chickens. More studies are needed to determine the potential mechanism of GA on broiler chickens.