The inability to engineer clinically relevant functional muscle tissue remains a major hurdle to successful skeletal muscle reconstructive procedures. This article describes an in vitro preconditioning protocol that improves the contractility of engineered skeletal muscle after implantation in vivo. Primary human muscle precursor cells (MPCs) were seeded onto collagen-based acellular tissue scaffolds and subjected to cyclic strain in a computer-controlled bioreactor system. Control constructs (static culture conditions) were run in parallel. Bioreactor preconditioning produced viable muscle tissue constructs with unidirectional orientation within 5 days, and in vitro-engineered constructs were capable of generating contractile responses after 3 weeks of bioreactor preconditioning. MPC-seeded constructs preconditioned in the bioreactor for 1 week were also implanted onto the latissimus dorsi muscle of athymic mice. Analysis of tissue constructs retrieved 1 to 4 weeks postimplantation showed that bioreactor-preconditioned constructs, but not statically cultured control tissues, generated tetanic and twitch contractile responses with a specific force of 1% and 10%, respectively, of that observed on native latissimus dorsi. To our knowledge, this is the largest force generated for tissue-engineered skeletal muscle on an acellular scaffold. This finding has important implications to the application of tissue engineering and regenerative medicine to skeletal muscle replacement and reconstruction.