Cancer development is a complex process governed by the interaction of several signaling pathways. The Hippo and PI3K/Akt pathways have been shown to play a critical role in controlling tissue growth involved in the regulation of cell proliferation. 3, 3'-diindolylmethane (DIM) is a natural compound that selectively kills cancer cells without causing toxicity to normal cells. This study aims to investigate whether DIM has an effect on the Hippo signaling pathway mediated via the PI3K/Akt signaling pathway in colon cancer cells. Our study provides new insights into the mechanisms of crosstalk between Hippo signaling and the Akt pathway controlling cell proliferation by PI3K inhibitor and DIM treatment in colon cancer cells. DIM strongly potentiates the lethality of LY294002 in HCT116 cells and inhibits proliferation of colon cancer cells via inactivation of Akt and YAP. Thus, DIM has dramatic therapeutic effects when it is combined with the PI3K inhibitor in the treatment of colon cancer cells. These findings highlight the potential usefulness of DIM and can help develop therapeutic strategies for the prevention and treatment of colon cancer.