We previously showed that divalent cations regulate alpha2beta1 integrin-mediated pancreatic cancer cell interactions with type I collagen in 2 dimensions (2D), including cell adhesion, migration, and proliferation. Presently, we examined divalent cation-dependent alpha2beta1 integrin-mediated pancreatic cancer cell adhesion and proliferation on type I collagen in a novel 3D in vitro model.Cell attachment, proliferation, and antibody inhibition assays on type I collagen in both 2D and 3D, and microscopy and immunoblotting were used for these studies.As in 2D, cell attachment on type I collagen in 3D is Mg-dependent and inhibited by Ca. Proliferation in 3D is also Mg-dependent, but maximal when Mg is present at concentrations that promote maximal cell adhesion and Ca is present at concentrations less than Mg. Immunoblotting studies demonstrate that the divalent cation-dependent changes in cell-cell adhesion observed on type I collagen in both 2D and 3D are associated with the changes in E-cadherin and beta-catenin expression. Antibody inhibition assays indicate further that the alpha2beta1 integrin specifically mediates proliferation on type I collagen in 3D under altered divalent cation conditions.Divalent cation shifts could activate alpha2beta1 integrin-mediated malignancy in the type I collagen-rich 3D tumor microenvironment of pancreatic cancer.