Regulatory dendritic cells (DCs) play an important role in maintaining peripheral tolerance or immune homeostasis. Our previous study demonstrated that mature DCs could be driven by splenic stroma to proliferate and differentiate into a novel subset of regulatory DCs (diffDCs) displaying a Th2-biased cytokine profile. However, the underlying mechanisms for the unique cytokine profile of diffDCs and how diffDCs regulate the innate and adaptive immunity in response to toll-like receptor (TLR) agonists remain unclear. Here, we report that unlike immature DCs, diffDCs secrete more interleukin 10 (IL-10) but little IL-12p70 in response to lipopolysaccharide (LPS) or other TLR agonists. Up-regulation of extracellular signal-regulated kinase (ERK1/2) activation was shown to be responsible for IL-10 preferential production, and suppression of p38 activation was for impaired IL-12p70 production in diffDCs. Interestingly, LPS treatment could not reverse the inhibitory effect of diffDCs on the proliferation of antigen-specific CD4+ T cells. However, diffDCs could activate natural killer (NK) cells through diffDC-derived IL-10, and even more markedly after stimulation of TLR agonists. These diffDC-activated NK cells could in turn kill surrounding diffDCs. Our results illuminate signal pathways for the unique cytokine profile of diffDCs, and diffDCs can exert their regulatory function even after inflammatory stimuli, thus reflecting one way for strict regulation of immune response.