Microglial activation has long been recognized as a hallmark of neuroinflammation. Recently, the bacillus Calmette-Guerin (BCG) vaccine has been reported to exert neuroprotective effects against several neurodegenerative disorders. Trehalose-6,6'-dibehenate (TDB) is a synthetic analogue of trehalose-6,6'-dimycolate (TDM, also known as the mycobacterial cord factor) and is a new adjuvant of tuberculosis subunit vaccine currently in clinical trials. Both TDM and TDB can activate macrophages and dendritic cells through binding to C-type lectin receptor Mincle; however, its action mechanism in microglia and their relationship with neuroinflammation are still unknown. In this article, we found that TDB inhibited LPS-induced M1 microglial polarization in primary microglia and BV-2 cells. However, TDB itself had no effects on IKK, p38, and JNK activities or cytokine expression. In contrast, TDB activated ERK1/2 through PLC-γ1/PKC signaling and in turn decreased LPS-induced NF-κB nuclear translocation. Furthermore, TDB-induced AMPK activation via PLC-γ1/calcium/CaMKKβ-dependent pathway and thereby enhanced M2 gene expressions. Interestingly, knocking out Mincle did not alter the anti-inflammatory and M2 polarization effects of TDB in microglia. Conditional media from LPS-stimulated microglial cells can induce in vitro neurotoxicity, and this action was attenuated by TDB. Using a mouse neuroinflammation model, we found that TDB suppressed LPS-induced M1 microglial activation and sickness behavior, but promoted M2 microglial polarization in both WT and Mincle-/- mice. Taken together, our results suggest that TDB can act independently of Mincle to inhibit LPS-induced inflammatory response through PLC-γ1/PKC/ERK signaling and promote microglial polarization towards M2 phenotype via PLC-γ1/calcium/CaMKKβ/AMPK pathway. Thus, TDB may be a promising therapeutic agent for the treatment of neuroinflammatory diseases.