This study aimed to compare the immune function and gut microbiota between double-layer caged and litter floor pen-raised broiler chickens. Eighty meaty male chicks were selected and divided into cage group and litter floor group, with 20 replicates in each group. The broilers were raised in the same chicken house. The rearing density of the two rearing systems was same. The broilers were sampled on days 13 and 34. The results showed that compared with the cage group, the litter floor broilers had worse growth performance (23.24% increase in feed conversion ratio) in the early stage; better slaughter performance at day 42; stronger peripheral immune function (including higher lysozyme activity, T-cell ratio, Th-cell ratio, Tc-cell ratio, CD4/CD8, IL-10, B-cell ratio, IgG and IgA levels; and spleen immune-related gene expression); and stronger intestinal immune function (including higher ileum CD80, AvBD2, Mucin2, NF-κB, IL-8, IFN-γ/IL-4, and IgA mRNA expression levels and ileal mucosa sIgA levels). Compared with the cage group, the alpha diversity of ileum microbiota of the litter floor broilers was higher, and the relative abundance levels of litter breeding bacteria (Facklamia, Globicatella, and Jeotgalicoccus) and potential pathogenic bacteria (Streptococcus and Staphylococcus) were higher (P < 0.05). Through Spearman correlation analysis, it was found that enriched microbes in the ileum of litter floor broilers were positively correlated with immune function. In summary, compared with cage broilers, litter floor broilers had more potential pathogenic bacteria and litter breeding bacteria in the ileum, which may be one of the important reasons for the stronger immune function status. IMPORTANCE In China, the three-dimensional rearing system (cage) for broilers has gradually become a trend. In production, it was found that the incidence of disease in broiler chickens raised in cage systems was significantly higher than that of ground litter. Given that broilers raised on ground litter systems may be exposed to more environmental microbes, it is important to understand whether the rearing environment affects the function and status of the host immune system by altering the gut microbiota. In this study, rearing environment-derived gut microbes associated with stronger immune function in ground litter broilers were provided, which will provide new insights into strategies to target gut microbes to promote immune function and status in broilers raised in cages.