This present study aimed to investigate the effects of rhamnolipids (RLS) on the growth performance, intestinal morphology, immune function, short-chain fatty acid content, and microflora community in broiler chickens challenged with lipopolysaccharides (LPS). A total of 450 broiler chickens were randomly allocated into three groups: basal diet with no supplement (NCO), basal diet with bacitracin (ANT), and basal diet with rhamnolipids (RLS). After 56 d of feeding, 20 healthy broilers were selected from each group, with half being intraperitoneally injected with lipopolysaccharides (LPS) and the other half with normal saline. Treatments with LPS were labelled LPS-NCO, LPS-ANT, and LPS-RLS, whereas treatments with normal saline were labelled NS-NCO, NS-ANT, and NS-RLS. LPS-challenged birds had lower jejunal villus height and higher crypt depth than unchallenged birds. LPS-RLS broilers had increased jejunal villus height and villus height/crypt depth ratio (V/C) but lower crypt depth than LPS-NCO. Dietary supplementation with RLS reduced the LPS-induced immunological stress. Compared with LPS-NCO, birds in LPS-RLS had lower concentrations of IL-1β, IL-6, and TNF-α. In LPS-challenged broilers, RLS and ANT increased the concentrations of IgA, IgM, and IgY compared with LPS-NCO. In LPS treatments, RLS enhanced the contents of acetic acid, butyrate, isobutyric acid, isovalerate, and valerate more than LPS-NCO birds. High-throughput sequencing indicated that RLS supplementation led to changes in the cecal microbial community of broilers. At the species level, Clostridium-sp-Marseille-p3244 and Slakia_eqcsolifaciens were more abundant in NS-RLS than in NS-NCO broilers. In summary, RLS improved the growth performance and relative abundance of cecal microbiota and reduced the LPS-induced immunological stress in broiler chickens.