Tendons are essential for transmitting muscle forces and maintaining joint stability, and their ability to adapt to mechanical tension is key for maintaining homeostasis. When tension is lost, tendons activate catabolic pathways that can lead to matrix breakdown and degeneration. This study examined how components of the cytoskeletal–nuclear mechanotransduction pathway regulate tendon contraction. Tail tendons from Myh9/10 and wild-type CD1 mice were cultured and treated with agents targeting specific parts of this pathway, including blebbistatin, TGF-β, and a FAK inhibitor. Myh9/10 deletion and blebbistatin treatment both significantly reduced tendon contractility, demonstrating that non-muscle myosin II is essential for generating contraction forces. In contrast, TGF-β increased contractility and upregulated mechanoactive genes (Acta2, Cyr61, Ctgf, Col1a1), while inhibitors suppressed their expression. These results show that tendon cells depend on the cytoskeletal–nuclear connection to sense mechanical cues and regulate both contraction and gene expression. Overall, this study highlights the importance of myosin in tendon contractility and provides insight into how disruptions in mechanotransduction may contribute to tendinopathy progression.