Clenbuterol, a β2-agonist, induces skeletal muscle hypertrophy and a shift from slow-oxidative to fast-glycolytic muscle fiber type profile. However, the cellular mechanisms of the effects of chronic clenbuterol administration on skeletal muscle are not completely understood. As the intracellular Ca2+ concentration must be finely regulated in many cellular processes, the aim of this study was to investigate the effects of chronic clenbuterol treatment on force, fatigue, intracellular calcium (Ca2+) homeostasis and Ca2+-dependent proteolysis in fast-twitch skeletal muscles (the extensor digitorum longus, EDL, muscle), as they are more sensitive to clenbuterol-induced hypertrophy. Male Wistar rats were chronically treated with 4 mg.kg-1 clenbuterol or saline vehicle (controls) for 21 days. Confocal microscopy was used to evaluate sarcoplasmic reticulum Ca2+ load, Ca2+-transient amplitude and Ca2+ spark properties. EDL muscles from clenbuterol-treated animals displayed hypertrophy, a shift from slow to fast fiber type profile and increased absolute force, while the relative force remained unchanged and resistance to fatigue decreased compared to control muscles from rats treated with saline vehicle. Compared to control animals, clenbuterol treatment decreased Ca2+-transient amplitude, Ca2+ spark amplitude and frequency and the sarcoplasmic reticulum Ca2+ load was markedly reduced. Conversely, calpain activity was increased by clenbuterol chronic treatment. These results indicate that chronic treatment with clenbuterol impairs Ca2+ homeostasis and this could contribute to the remodeling and functional impairment of fast-twitch skeletal muscle.