The mechanism(s) underlying eccentric damage to skeletal muscle cytoskeleton remain unclear. We examined the role of Ca²⁺ influx and subsequent calpain activation in eccentric damage to cytoskeletal proteins. Eccentric muscle damage was induced by stretching isolated mouse muscles by 20% of the optimal length in a series of 10 tetani. Muscle force and immunostaining of the cytoskeletal proteins desmin, dystrophin and titin, were measured at 5, 15, 30 and 60 min after eccentric contractions and compared with the control group that was subjected to 10 isometric contractions. A Ca²⁺-free solution and leupeptin (100 µM), a calpain inhibitor, were applied to explore the role of Ca²⁺ and calpain, respectively, in eccentric muscle damage. After eccentric contractions, decreases in desmin and dystrophin immunostaining were apparent after 5 min that accelerated over the next 60 min. Increased titin immunostaining, thought to indicate damage to titin, was evident 10 min after stretch, and fibronectin entry, indicating membrane disruption, was evident 20 min after stretch. These markers of damage also increased in a time-dependent manner. Muscle force was reduced immediately after stretch and continued to fall reaching 56 ± 2% after 60 min. Reducing extracellular calcium to zero or applying leupeptin minimized the changes in immunostaining of cytoskeletal proteins, reduced membrane disruption and improved the tetanic force. These results suggest that the cytoskeletal damage and membrane disruption were mediated primarily by increased Ca²⁺ influx into muscle cells and subsequent activation of calpain.