Cancer-associated fibroblasts (CAFs) play critical roles in the tumor progression. However, it remains unclear how cancer cells migrate in the three-dimensional (3D) matrix of cancer tissues and how CAFs support the cancer invasion. Here we propose a novel mechanism of fibroblast-dependent cancer cell invasion in the 3D collagen matrix. Human cancer cell lines from the pancreas (Panc-1), lung (A549) and some other organs actively adhered to normal fibroblasts and primary lung CAFs in cultures. To show its significance in tumor invasion, we designed a new invasion assay in which homogeneous microspheroids consisting of cancer cells and fibroblasts were embedded into collagen gel. Time-lapse experiments showed that cancer cells adhered to and quickly migrated on the long protrusions of fibroblasts in the 3D collagen matrix. Fibroblast-free cancer cells poorly invaded the matrix. Experiments with function-blocking antibodies, siRNAs, and immunocytochemistry demonstrated that cancer cells adhered to fibroblasts through integrin α5β1-mediated binding to fibronectin on the surface of fibroblasts. Immunochemical analyses of the co-cultures and lung cancers suggested that cancer cells could acquire the migratory force by the fibronectin/integrin signaling. Our results also revealed that the fibroblast-bound fibronectin was a preferential substrate for cancer cells to migrate in the collagen matrix.