The ability of dendritic cells (DCs) to initiate and regulate adaptive immune responses is fundamental for maintaining immune homeostasis upon exposure to self or foreign antigens. The immune regulatory function of DCs is strictly controlled by their distribution as well as by cytokines, chemokines, and transcriptional programming. These factors work in conjunction to determine whether DCs exert an immunosuppressive or immune-activating function. Therefore, understanding the molecular signals involved in DC-dependent immunoregulation is crucial in providing insight into the generation of organismal immunity and revealing potential clinical applications of DCs. Considering the many breakthroughs in DC research in recent years, in this review we focused on three basic lines of research directly related to the biological functions of DCs and summarized new immunotherapeutic strategies involving DCs. First, we reviewed recent findings on DC subsets and identified lineage-restricted transcription factors that guide the development of different DC subsets. Second, we discussed the recognition and processing of antigens by DCs through pattern recognition receptors, endogenous/exogenous pathways, and the presentation of antigens through peptide/major histocompatibility complexes. Third, we reviewed how interactions between DCs and T cells coordinate immune homeostasisin vivo viamultiple pathways. Finally, we summarized the application of DC-based immunotherapy for autoimmune diseases and tumors and highlighted potential research prospects for immunotherapy that targets DCs. This review provides a useful resource to better understand the immunomodulatory signals involved in different subsets of DCs and the manipulation of these immune signals can facilitate DC-based immunotherapy.