Metastatic cancer attributes to a major cause of cancer death. In this pioneer study, we aimed to investigate how Antrodia cinnamomea (A. cinnamomea), indigenous to Taiwan, affects migration ability of highly metastatic human adenocarcinoma lung cancer cells CL1-5. Our result demonstrated that noncytotoxic ethanol extract of fruiting bodies of A. cinnamomea (EEAC) exhibited a dose-dependent inhibitory effect on motility and migration of the highly metastatic CL1-5 cells. Results of a gelatin zymography assay illustrated that A. cinnamomea repressed the activities of matrix metalloproteinase- (MMP-) 2 and 9 in a dose-dependent manner. A. cinnamomea administration decreased MMP-9 and MMP-2 protein expressions from Western blotting assay, whereas the expression of the tissue inhibitors of MMP (TIMP-1 and TIMP-2) increased. Additional study disclosed that A. cinnamomea suppressed FAK, ERK1/2, p38, AKT, and JNK1/2 phosphorylation, and also PI3K and Rac-1 were found decreased. Further, treatment of CL1-5 cells with inhibitors specific for PI3K (LY294002), ERK1/2 (PD98059), JNK (SP600125), and p38 MAPK (SB203580) decreased the expression of MMP-2 and MMP-9. Taken together, EEAC induced FAK phosphorylation and exhibited its antimigration activities via the PI3K/AKT and MAPK signalings in CL1-5 cells. This is the pioneer study verifying the antimigration activity of A. cinnamomea against human lung adenocarcinoma CL1-5 cancer cells [corrected].