Abstract Background Previous studies suggest that increased ubiquitin-proteasome–dependent protein breakdown in various muscle-wasting conditions may at least in part be mediated by increased cellular calcium levels. The role of calcium in the regulation of proteasome activity, however, is not well understood. Methods We treated cultured L6 myotubes with the calcium ionophore A23187 or thapsigargin, substances that increase intracellular calcium levels through different mechanisms, and measured proteasome activity by determining the degradation of the fluorogenic substrate LLVY-AMC. Results Treatment of the myotubes with A23187 or thapsigargin resulted in a dose- and time-dependent increase in proteasome activity. When the myotubes were treated with metabolic inhibitors, results suggested that the A23187- and thapsigargin-induced activation of proteasome activity was at least in part regulated by calmodulin, calcium calmodulin–dependent kinase II, caspases, and calpains. Conclusions The present observations support a role of calcium in the regulation of proteasome-dependent protein breakdown in skeletal muscle and may explain why muscle wasting was reduced by calcium antagonists in previous studies.