Abstract: OBJECTIVE To investigate the anti-cancer effects of Ganoderma lucidum water extract(GLWE) based on the two-dimensional(2D) and three-dimensional(3D) culture of colorectal cancer HCT116 cells.METHODS The content of Ganoderma lucidum polysaccharide(GLP), Ganoderma triterpenes and sterols in GLWE were determined by UV-visible spectrophotometry, and the composition and proportion of water-soluble monosaccharide in GLP were determined by HPLC. An 3D culture model of HCT116 cells in vitro was established using Matrigel as the matrix material, and the anti-cancer and adjuvant anti-cancer activities of GLWE were evaluated. Cell viability was detected by CCK-8 assay. mRNA expression level of the cells was analyzed by Real-time PCR. Western blotting was used to detect cell protein expression level. RESULTS The contents of GLP and Ganoderma triterpenes(contained sterols) in GLWE were 20.92% and 7.18%, respectively, and GLP was mainly comprised of mannose, glucuronic acid, glucose, galactose, D-xylose and L-fucose. GLWE and 5-fluorouracil(5-FU) inhibited the proliferation of both 2D and 3D cultured HCT116 cells in a dose dependent manner after incubation for 48 h. However, compared with the 2D culture, the expression of E-cadherin and Integrin β1 mRNA was significantly increased, and the sensitivity to GLWE and 5-FU was reduced in 3D cultured HCT116 cell. GLWE could not only reduce the expression of CDK2, CDK4 and Bcl-2 proteins, increase the expression of p21, p27, cleaved caspase-3 and cleaved PARP proteins, but also significantly reduce the expression of E-cadherin and Integrin β1 mRNA, and enhance the anti-cancer activity of 5-FU in 3D cultured HCT116 cell. CONCLUSION GLWE significantly inhibit the proliferation of 3D-cultured HCT116 cells by inhibiting cell cycle and inducing apoptosis, and enhanced the anti-cancer activity of 5-FU by inhibiting the expression of E-cadherin and Integrin β1 mRNA.