Antrodia camphorata (AC) exhibits potential for engendering cell-cycle arrest as well as prompting apoptosis and metastasis inhibition in triple-negative breast cancer (TNBC) cells. We performed the current study to explore the anti-epithelial-to-mesenchymal transition (EMT) properties of fermented AC broth in TNBC cells. Our results illustrated that noncytotoxic concentrations of AC (20-60 μg/ml) reversed the morphological changes (fibroblastic-to-epithelial phenotype) as well as the EMT by upregulating the observed E-cadherin expression. Furthermore, we discovered treatment with AC substantially inhibit the Twist expression in human TNBC (MDA-MB-231) cells as well as in those that were transfected with Twist. In addition, we determined AC to decrease the observed Wnt/β-catenin nuclear translocation through a pathway determined to be dependent on GSK3β. Notably, AC treatment consistently inhibited the EMT by downregulating mesenchymal marker proteins like N-cadherin, vimentin, Snail, ZEB-1, and fibronectin; at that same time upregulating epithelial marker proteins like occludin and ZO-1. Bioluminescence imaging that was executed in vivo demonstrated AC substantially suppressed breast cancer metastasis to the lungs. Notably, we found that western blot analysis confirmed that AC decreased lung metastasis as demonstrated by upregulation of E-cadherin expression in biopsied lung tissue. Together with our results support the anti-EMT activity of AC, indicating AC as having the potential for acting as an anticancer agent for the treatment of human TNBC treatment.