Caspase recruitment domain 6 (CARD6) was initially implicated in the immune system and oncogenesis, which has also been emerged to play an important role in cardio-metabolic diseases. Nevertheless, the potential role of CARD6 on macrophage activation remains unknown. In the present study, we observed a decreased CARD6 expression in bone marrow derived macrophages (BMDMs) and mouse peritoneal macrophages (MPMs) isolated from ApoE deficiency mice and administrated with OX-LDL, which were tested by RT-PCR and western bolt analysis. Moreover, the immunofluorescence co-staining revealed that a weaker immunoreactivity of CARD6 was found and primary located in cytoplasm of macrophages induced by OX-LDL. Phenotypically, loss-of-function of CARD6 dramatically increased pro-inflammatory M1 macrophage but decreased resolving M2 macrophage markers expression. Additionally, CARD6 knockdown significantly promoted cholesterol uptake but attenuated cholesterol efflux, which lead to increased foam cell formation. Mechanistically, a downregulated AMP-activated protein kinase (AMPK) expression was required for the promoted effect of CARD6 knockdown on macrophage activation. Taken together, these results suggest that CARD6 protects against macrophage activation partially through activation of AMPK-dependent mechanism. (C) 2020 Published by Elsevier Inc.