3-Methyladenine (3-MA) is one of the most commonly used inhibitors in autophagy research today. However, rather than inhibiting class III PI3K that is involved in autophagy suppression, 3-MA might also interfere with class I PI3K and consequently augment autophagy flux. In this study, we aim to get a thorough understanding on the action mechanisms of 3-MA in TLR4-mediated inflammatory responses in RAW264.7 macrophages and, moreover, to decipher the action of 3-MA in modulation of autophagy. We found that 3-MA could enhance LPS-induced NF-KB activation and production of TNF-α, inducible NO synthase (iNOS), cyclooxygenase-2, IL-10, and IL-12. In contrast, 3-MA suppressed LPS-induced IFN-P production and STAT signaling. Studies revealed that 3-MA can, through inhibition of Akt as a result of class I PI3K interference, positively regulate p38, JNK, and p65, but negatively regulate TANK-binding kinase 1 and IFN regulatory factor 3 mediated by TLR4. As glycogen synthase kinase 3β (GSK3β) is an important Akt substrate, we further explored its involvement in the actions of 3-MA. 3-MA was found to enhance LPS-induced NF-KB activation, iNOS, and pro-IL-ip expression, and these actions were reversed by either GSK3fJ inhibitors or small interfering GSK3p. Lastly, we demonstrated that 3-MA acts as an autophagy inducer in RAW264.7 macro-phages, but the stimulating effects on NF-KB activation and iNOS and cyclooxygenase-2 expression were not affected in LPS-stimulated macrophages with small interfering autophagy protein-5 treatment. These results not only shed new light on the action mechanisms of 3-MA to differentially regulate inflammatory outcomes derived from TLR4-mediated MyD88 and Toll/IL-IR domain-containing adapter inducing IFN-β pathways, but also highlight the necessity to check autophagy status upon taking 3-MA as a general autophagy inhibitor. [ABSTRACT FROM AUTHOR]