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Despite advancements in surgical techniques and materials for rotator cuff repair procedures, primary repair failures remain common. This study examines the use of electrochemically aligned collagen (ELAC) threads woven into biotextile scaffolds as grafts to repair critical infraspinatus tendon defects in New Zealand White rabbits. Three surgical treatment groups were evaluated: rabbits undergoing direct repair as operative controls, rabbits receiving ELAC scaffolds alone, and rabbits treated with mesenchymal stem cell (MSC)‐seeded ELAC scaffolds. In each animal, the intact, contralateral infraspinatus served as an internal positive control. Tendon–bone constructs were harvested after 3 months in vivo and outcome measures included biomechanical testing, histological staining, and immunohistochemical staining. Biomechanical testing revealed that maximum load‐bearing capacity was comparable between all groups, while MSC‐seeded scaffold repairs exhibited increased stiffness relative to non‐seeded scaffold repairs. Histological staining revealed robust collagen deposition around ELAC fibers and increased cellularity within the continuum of woven scaffolds as compared to native tendon. Immunohistochemical staining revealed presence of collagens I and III in all groups, but procollagen I and the tendon‐specific marker tenomodulin were only observed in seeded and non‐seeded ELAC scaffold repairs. Findings of this pilot study warrant continued investigation of ELAC biotextile scaffolds for repair of critically‐sized rotator cuff tendon defects. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1864–1876, 2019. [ABSTRACT FROM AUTHOR]