Dendritic cells (DCs) are potent antigen-presenting cells derived from hematopoietic progenitor cells and circulating monocytes. To investigate the role of microRNAs (miRNAs) during DC differentiation, maturation, and function, we profiled miRNA expression in human monocytes, immature DCs (imDCs), and mature DCs (mDCs). Stage-specific, differential expression of 27 miRNAs was found during monocyte differentiation into imDCs and mDCs. Among them, decreased miR-221 and increased miR-155 expression correlated with p27 kip1 accumulation in DCs. Silencing of miR-221 or overexpressing of miR-155 in DCs resulted in p27 kip1 protein increase and DC apoptosis. Moreover, mDCs from miR-155 -/- mice were less apoptotic than those from wild-type mice. Silencing of miR-155 expression had little effect on DC maturation but reduced IL-12p70 production, whereas miR-155 overexpression in mDCs enhanced IL-12p70 production. Kip1 ubiquitination-promoting complex 1, suppressor of cytokine signaling 1, and CD115 (M-CSFR) were functional targets of miR-155. Furthermore, we provide evidence that miR-155 indirectly regulated p27 kip1 protein level by targeting Kip1 ubiquitination-promoting complex 1. Thus, our study uncovered miRNA signatures during monocyte differentiation into DCs and the new regulatory role of miR-221 and miR-155 in DC apoptosis and IL-12p70 production.