Plasmacytoid dendritic cells (pDCs) at tumor sites are often tolerogenic. Although pDCs initiate innate and adaptive immunity upon Toll-like receptor (TLR) triggering by pathogens, TLR-independent signals may be responsible for pDC activation and immune suppression in the tumor inflammatory environment. To identify molecules that are potentially involved in alternative pDC activation, we explored the expression and function of lymphocyte activation gene 3 (LAG-3) in human pDCs. In this report, we showed the expression of LAG-3 on the cell surface of a subset of circulating human pDCs. LAG-3+ pDCs exhibited a partially mature phenotype and were enriched at tumor sites in samples from melanoma patients. We found that LAG-3 interacted with major histocompatibility complex class II (MHC-II) to induce TLR-independent activation of pDCs with limited IFNα and enhanced IL-6 production. This in vitro cytokine profile of LAG-3-activated pDCs paralleled that of tumor-associated pDCs analyzed ex vivo. By confocal microscopy, LAG-3+ pDCs detected in melanoma-invaded lymph nodes (LNs) stained positive for IL-6 and preferentially localized near melanoma cells. These results suggest that LAG-3-mediated activation of pDCs takes place in vivo at tumor sites, and it is in part responsible for directing an immune-suppressive environment.