Mucosal dendritic cells (DCs) play a key role in discriminating between dietary antigens, commensal microflora and pathogens but little is known regarding age-related changes in mucosal DC populations. We analyzed lymphoid and myeloid populations within the epithelium and lamina propria (LP) of the ileum and jejunum of weaned calves (6 months old) and compared their frequency and distribution with newborn calves (3-5 weeks old). CD4, CD8 and γδ TcR T cells and CD11c(Hi)MHC Class II(+) myeloid cell frequency were significantly different when comparing ileum and jejunum of weaned calves. In particular, the number of CD8 and γδ TcR T cells, and CD11c(Hi)CD14(+) macrophages was significantly greater in the ileum but CD11c(+) and CD11b(+) myeloid cell distribution was similar throughout the mucosal epithelium of the small intestine. Furthermore, significant age-related changes were apparent when comparing the frequency and abundance of mucosal leukocyte subpopulations in newborn and weaned calves. Total mucosal leukocytes (CD45(+)) increased significantly with age in both ileum and jejunum and much of this increase was attributed to mucosal T cells (CD3(+)). In particular, CD4 T cells and NK cells increased significantly in the jejunum and CD8, and γδ TcR T cells increased significantly with age throughout the small intestine. In contrast, CD11c(Hi)MHC Class II(+) myeloid cells remained numerically unchanged with age but DCs (CD13(+), CD26(+), CD205(+)) were enriched and macrophages (CD14(+), CD172a(+)) were depleted in older animals. Therefore, regional differences between ileal and jejunal mucosal leukocytes changed with age and there was also a marked age-dependent change in the composition of mucosal myeloid cells. These observations have significant implications for host responses to both pathogens and commensal microflora.