Skeletal muscle adapts to endurance exercise with an increase in mitochondria. Muscle contractions generate numerous potential signals. To determine which of these stimulates mitochondrial biogenesis, we are using L6 myotubes. Using this model we have found that raising cytosolic Ca²⁺ induces an increase in mitochondria. In this study, we tested the hypothesis that raising cytosolic Ca²⁺ in L6 myotubes induces increased expression of PGC‐1, NRF‐1, NRF‐2, and mtTFA, factors that have been implicated in mitochondrial biogenesis and in the adaptation of muscle to exercise. Raising cytosolic Ca²⁺ by exposing L6 myotubes to caffeine for 5 h induced significant increases in PGC‐1 and mtTFA protein expression and in NRF‐1 and NRF‐2 binding to DNA. These adaptations were prevented by dantrolene, which blocks Ca²⁺ release from the SR. Exposure of L6 myotubes to caffeine for 5 h per day for 5 days induced significant increases in mitochondrial marker enzyme proteins. Our results show that the adaptive response of L6 myotubes to an increase in cytosolic Ca²⁺ mimics the stimulation of mitochondrial biogenesis by exercise. They support the hypothesis that an increase in cytosolic Ca²⁺ is one of the signals that mediate increased mitochondrial biogenesis in muscle.