Medicinal mushroom Ganoderma lucidum is one of the most esteemed natural products that have been used in the traditional Chinese medicine. In this article, we demonstrate that G. lucidum triterpene extract (GLT) suppresses proliferation of human colon cancer cells HT-29 and inhibits tumor growth in a xenograft model of colon cancer. These effects of GLT are associated with the cell cycle arrest at G0/G1 and the induction of the programmed cell death Type II-autophagy in colon cancer cells. Here, we show that GLT induces formation of autophagic vacuoles and upregulates expression of Beclin-1 (1.3-fold increase) and LC-3 (7.3-fold increase) proteins in colon cancer cells and in tumors in a xenograft model (Beclin-1, 3.9-fold increase; LC-3, 1.9-fold increase). Autophagy is mediated through the inhibition of p38 mitogen-activated protein kinase (p38 MAPK) because p38 MAPK inhibitor, SB202190, induces autophagy and expression of Beclin-1 (1.2-fold increase) and LC-3 (7.4-fold increase), and GLT suppresses phosphorylation of p38 MAPK ( approximately 60% inhibition) in colon cancer cells. Taken together, our data demonstrate a novel mechanism responsible for the inhibition of colon cancer cells by G. lucidum and suggest GLT as natural product for the treatment of colon cancer.