Tumor metastasis and invasiveness are research hotspots in tumor therapy. Previous research demonstrated that cells expressing high level of CD36 might initiate metastasis in certain human oral carcinomas cell lines. Multicellular spheroids (MCS) culture technique has been extensively studied both in fundamental and applied research. In our previous research, we invented novel 3D anisotropic magnetic hydrogels (AMHs) which were composed of magnetic nanoparticle assemblies and biocompatible hydrogels. We further applied them as cell culture substrate in vitro. In this study, HT-29 cells were cultured on the 3D AMHs, and cell viability and protein expression were investigated. Real time-PCR and Western-blot were carried out to determine the expression of a series of proteins associated with metastasis. Results revealed that cell growth was significantly decreased in AMHs group compared with controls. Meanwhile, microenvironment greatly affected the protein expression of the cells within. The CD36 expression in the AMHs group was ∼1.5-fold higher than in the control group. Moreover, The Western blot analysis demonstrated that the level of CD36 was higher in the AMHs group than in the 2D group. Besides, it was found that the expressions of N-cadherin and Snail in the HT29 cells were highly improved in the AMHs. As for the expression of ß-catenin and E-cadherin, an obvious decrease was seen in the AMHs than in the 2D group. Our results demonstrated that the elevated expression of CD36 in the HT29 cells, which were cultured on 3D AMHs, was associated with epithelial-mesenchymal transition (EMT) induction. The results further indicated the importance of 3D AMHs cell culture platform in vitro.