AbstractStromal collagen surrounding a solid tumor tends to present as dense, thick bundles. The collagen bundles are remodeled during tumor progression: first tangential to the tumor boundary (indicating growth) and later perpendicular to the tumor boundary (indicating likely metastasis). Current reconstituted-collagen in vitro tumor models are unable to recapitulate the in vivo structural features of collagen bundling and alignment. Here, we present a rapid yet simple procedure to fabricate collagen bundles with an average thickness of 9 μm, compared to the reticular dense collagen nanofiber (∼900 nm-diameter, on average) prepared using common protocols. The versatility of the collagen bundles was demonstrated with their incorporation into two in vitro models where the thickness and alignment of the collagen bundles resembled the various in vivo arrangements. First, collagen bundles aligned by a microfluidic device elicited cancer cell contact guidance and enhanced their directional migration. Second, the presence of the collagen bundles in a bio-inert agarose hydrogel was shown to provide a highway for cancer cell invasion. The unique structural features of the collagen bundles advance the physiological relevance of in vitro collagen-based tumor models for accurately capturing cancer cell-stroma interactions.