Activation of microglia triggers a robust secretion of pro-inflammatory cytokines, which are implicated in the pathogenesis of central nervous system tuberculosis (CNS-TB). Trehalose-6,6’-dimycolate (TDM), the mycobacterial cord factor, and its synthetic analogue trehalose-6,6’-dibehenate (TDB) bind to C-type lectin receptor Mincle, and are shown to activate macrophages and dendritic cells through the Syk/Card9 pathway. However, until now it is totally unknown the biological functions of Mincle in microglia. In this article, we reported that TDB inhibited LPS-induced inflammatory response and promoted microglial M2 polarization in primary microglia and BV-2 cells. However, TDB alone did not result in significant production of cytokines in microglial cells. Furthermore, TDB activated ERK1/2 in LPS-stimulated cells, but it had no effects on IKK, p38 or JNK activity. Surprisingly, TDB suppressed LPS-induced NF-κB nuclear translocation via ERK activation. In addition, TDB caused PLC-γ1 and PKC activation in BV-2 cells, and TDB-mediated ERK activation could be attenuated by PLC-γ1 and PKC inhibitors. Furthermore, TDB induced AMPK activation through PLC-γ1/calcium/CaMKKβ-dependent cascade, leading to enhance M2 phenotype gene expression in LPS-activated BV-2 cells. Importantly, blocking or knocking out Mincle did not abrogate TDB-meditated anti-inflammatory responses and phenotypic polarization in BV-2 cells and primary microglia. Conditional medium from LPS-stimulated primary microglia can induce neurotoxicity, and this action was attenuated by the presence of TDB, and was independent of Mincle. In a murine CNS-inflammation model, we found that TDB significantly reduced LPS-induced microglial activation, expression of pro-inflammatory cytokines and sickness behaviour in both wild type and Mincle-/- mice. Together, these data suggest that TDB exerts the Mincle-independent mechanism in microglia to attenuate LPS-induced inflammatory response via PLC-γ1/PKC/ERK pathway, and also polarizes M2 phenotype of microglia via PLC-γ1/calcium/CaMKKβ/AMPK pathway. Thus, TDB may be exploited against CNS-TB infection and neuroinflammatory diseases.