Dendritic cell (DC) can be stimulated by both exogenous pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and endogenous damage-associated molecular patterns (DAMPs) such as high mobility group box-1 protein (HMGB1). MicroRNAs (miRNAs) act as post-transcriptional fine tuners of mRNA. Studies have focused mostly on the potential role of miRNAs in DCs maturation triggered by PAMPs, especially LPS, however, little is known about the regulatory mechanism underlying the effects of miRNAs in DC maturation mediated by DAMPs, including HMGB1. Here, we first profiled a miRNA microarray of DCs stimulated by HMGB1 and determined that the up-regulated miRNA miR-181a-5p may act as a regulatory miRNA in these cells. Computational algorithms predicted TNF-α 3′UTR to be targeted by miR-181a-5p, which was confirmed by the experiments involving luciferase reporters. In addition, we found that TNF-α mRNA was down-regulated by miR-181a-5p mimic, and significantly up-regulated by miR-181a-5p inhibitor. Taken together, we identified miR-181a-5p a negative regulator in HMGB1-induced immune responses by targeting TNF-α mRNA in DCs. Moreover, we suggested that miR-181a-5p may play a role in regulating DC responses to HMGB1 and serve as evidence indicating that novel therapies targeting miRNAs may be useful for treating immune dysfunction in the setting of sepsis.