Well-aligned collagen fiber scaffolds are considered promising candidates for tendon tissue engineering in terms of their biomimetic chemical composition and topographic structure. Insoluble collagen fibers are more suitable for the preparation of scaffolds than soluble collagens due to their more approximate self-assembly and mechanical properties to native collagen ECMs. In this work, we employed counter-rotating extrusion technology for the first time to fabricate an aligned (CM, orientation angle 0°-15°) and a randomly-oriented (CM, orientation angle -60°-60°) collagen membrane from insoluble collagens. CM had a tensile strength comparable with native rat Achilles tendon (18.45 ± 0.91 MPa vs. 22.32 ± 2.48 MPa). Thus, CM represents a scaffold that is biomimetic of native tendon tissues in chemical composition, alignment, and mechanical properties. To verify the feasibility of CMs in tendon tissue engineering, we investigated the in vitro tenogenic differentiation of rBMSCs on CMs and the in vivo tendon regeneration using a rat Achilles tendon defect model. Detection of the tendon-related genes and proteins revealed that CM can promote significantly higher tenogenic differentiation of rBMSCs than CM, by inducing an elongated cell shape along the fibers. The in-situ tendon repair study further confirmed that CM-BMSCs can produce a comparable healing quality to the autogenous tendon. Overall, our results verify the feasibility of the counter-rotating extrusion technology in fabricating biomimetic collagen scaffolds and provide a promising scaffold for tendon tissue regeneration.