Musculoskeletal injuries in equine athletes represent one of the leading causes for sport-induced and career-ending injuries due to slow or incomplete tendon repair creating clinical challenges in functional repair and effective rehabilitation. As ideal strategies for tendon repair are being developed, testing these treatments begins with models. Three-dimensional (3D) tendon constructs provide an in vitro model highly representative of in vivo tendon. For some model species, protocols for cell seeding have been established, yet this is not the case for horse cells. Using a mouse 3D construct protocol with horse superficial digital flexor tendon (SDFT) cells led us to consider the need for protocol optimization for the horse cells. High cellularity in a construct could be advantageous due to the greater amount of cellular machinery for collagen production; however, more cells may produce spatial constraints thereby limiting area for collagen deposition. A balance between cell number and spatial constraint is necessary. Additionally, seeding technique – either by spreading the matrix or localizing – may affect mechanical or material properties due to differences in fibrinogen processing, collagen production and alignment. Biochemical, collagen content, and fibril ultrastructure organization analysis conducted on varying cell seeding concentrations (100,000, 300,000, and 500,000) showed that although not significant 300,000 cells per construct produced a more functionally and structurally reproducible construct. Assessing biomechanical and collagen content analysis for two seeding methods – local and spread – the localized method proved to be statistically stronger and more representative of in vivo tendon. Collectively, our findings show evidence that although cell seeding number does not ultimately affect the construct, the cell seeding method for three-dimensional constructs does affect the overall structure and function of the model tendon.