Mechanical force via muscle contraction is critical for the development and maturation of tendons and their integration with the adjacent bone through a specialized tissue known as an attachment unit. The mechanisms by which applied loads regulate tendon and attachment unit biology are not well understood, contributing to poor clinical outcomes for tendon pathologies. Actomyosin contractility, powered by non-muscle myosin (NM-II), generates contractile forces and transduces signals from applied loads to the nucleus, supporting critical cellular functions such as division, migration, mechanosignaling, and cytoskeletal organization. Given the importance of loading on tendon biology and NM-II’s central role in regulating cytoskeletal tension, we hypothesized that NM-II-mediated actomyosin contractility is critical to the formation and maintenance of tendons and their attachments to bone. To test this hypothesis, we generated cell-lineage specific transgenic knockout mice to specifically delete NM-II in Prrx1 (limb mesenchyme) or Scx (tenogenic) lineage cells. In Chapter 2, we found that deletion of the predominant NM-II isoforms, NM-IIA (Myh9 gene) and NMII-B (Myh10 gene) affected both the formation and maturation of the attachment unit. In particular, the initial formation of the deltoid tuberosity (DT) attachment unit failed in limb mesenchyme lacking one copy of Myh9 and both copies of Myh10. Even in genotypes where the DT formed, there were notable alterations in both the size and shape of this tissue. We also identified a cell-lineage specific role for NM-II in regulating DT maturation, as deletion of both Myh9 and Myh10 in Scx-lineage cells (“Myosin dKO”) similarly affected DT morphology. In Chapter 3, we assessed the role of NM-II in the tendon midsubstance and found that NM-II-mediated contractility is critical for proper tendon growth and maintenance. In Myosin dKO tendons, lateral tendon growth was significantly reduced, corresponding with a decrease in collagen fibril diameter. This deficit in growth coincided with an aberrant actin cytoskeleton which ultimately resulted in tendon degeneration, as P112 Myosin dKO tendons exhibited increased proteoglycan content, matrix disorganization, ectopic mineral, and decreased mechanical properties. Collectively, this work establishes NM-II as a key contributor to tendon and attachment unit development and maturation by regulating cell organization and matrix assembly.