Lactobacillus species are typical members of gut microflora that immunomodulatory effects and can regulate a variety of immune cells, such as dendritic cells (DCs). Notably, DCs possess the unique ability to initiate primary immune responses. Notably, DCs possess the unique ability to initiate primary immune responses. In this study, we investigated the effects of Lactobacillus johnsonii (L. johnsonii) on the maturation and activation of chicken bone marrow-derived dendritic cells (chBM-DCs). The chBM-DCs generated from chicken bone marrow monocytes were stimulated using lethally irradiated L. johnsonii. L. johnsonii-stimulated chBM-DCs upregulated the expression of major histocompatibility complex class II (MHC-II), CD40, and CD86, decreased phagocytosis, and increased the ability to induce the proliferation of allogeneic T cells, which displayed a mature phenotype and function. Upon maturation with L. johnsonii, the expression of Th1-type cytokines [interleukin (IL)-12, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α)], a Th2-type cytokine (IL-10), pro-inflammatory cytokines (IL-1β and IL-6), and chemokines (CXCLi1 and CXCLi2) greatly increased; however, a high expression of IL-10 was only observed at mid-late time points for chBM-DCs stimulated with high doses of L. johnsonii. Moreover, L. johnsonii upregulated the mRNA levels of TLR2 and TLR5. These results reveal that L. johnsonii plays a potentially important role in modulating the immunological functions of chBM-DCs, suggesting that it influences and mediates immune responses in vitro.Copyright © 2020 Elsevier Ltd. All rights reserved.