Mycoplasma hominis lacks a cell wall and lipoproteins anchored to the extracellular side of the plasma membrane are in direct contact with the host components. A Triton X-114 extract of M. hominis enriched with lipoproteins was shown to stimulate the production of IL-23 by human dendritic cells (hDCs). The inflammasome activation of the host cell has never been reported upon M. hominis infection. We studied here the interaction between M. hominis PG21 and hDCs by analyzing both the inflammation-inducing mycoplasmal lipoproteins and the inflammasome activation of the host cell.IL-23-inducing lipoproteins were determined using a sequential extraction strategy with two non-denaturing detergents, Sarkosyl and Triton X-114, followed by SDS-PAGE separation and mass spectrometry identification. The activation of the hDC inflammasome was assessed using PCR array and ELISA.We defined a list of 24 lipoproteins that could induce the secretion of IL-23 by hDCs, 5 with a molecular weight between 20 and 35 kDa and 19 with a molecular weight between 40 and 100 kDa. Among them, the MHO_4720 lipoprotein was identified as potentially bioactive and a synthetic lipopeptide corresponding to the N-terminal part of the lipoprotein was subsequently shown to induce IL-23 release by hDCs. Regarding the hDC innate immune response, inflammasome activation with caspase-dependent production of IL-1β was observed. After 24 h of co-incubation of hDCs with M. hominis, down-regulation of the NLRP3-encoding gene and of the adaptor PYCARD-encoding gene was noticed. Overall, this study provides insight into both protagonists of the interaction, M. hominis and hDCs.IMPORTANCEMycoplasma hominis is a human urogenital pathogen involved in gynecologic and opportunistic infections. M. hominis lacks a cell wall and its membrane contain many lipoproteins that are anchored to the extracellular side of the plasma membrane. In the present study, we focused on the interaction between M. hominis and human dendritic cells, and examined both sides of the interaction, the mycoplasmal lipoproteins involved in the activation of the host cell and the immune response of the cell. On the mycoplasmal side, we showed for the first time that M. hominis lipoproteins with high molecular weight were potentially bioactive. On the cell side, we reported an activation of the inflammasome, which is involved in the innate immune response.