The systemic effects of physical activity are mediated by the release of IL-6 and other myokines from contracting muscle. Although the release of IL-6 from muscle has been extensively studied, the information on the cellular mechanisms is fragmentary and scarce, especially regarding the role of Ca²⁺ signals. The aim of this study was to characterize the role of the main components of Ca²⁺ signals in human skeletal muscle cells during IL-6 secretion stimulated by the Ca²⁺ mobilizing agonist ATP. Primary cultures were prepared from surgical samples, fluorescence microscopy was used to evaluate the Ca²⁺ signals and the stimulated release of IL-6 into the medium was determined using ELISA. Intracellular calcium chelator Bapta, low extracellular calcium and the Ca²⁺ channels blocker La³⁺ reduced the ATP-stimulated, but not the basal secretion. Secretion was inhibited by blockers of L-type (nifedipine, verapamil), T-type (NNC55-0396) and Orai1 (Synta66) Ca²⁺ channels and by silencing Orai1 expression. The same effect was achieved with inhibitors of ryanodine receptors (ryanodine, dantrolene) and IP3 receptors (xestospongin C, 2-APB, caffeine). Inhibitors of calmodulin (calmidazolium) and calcineurin (FK506) also decreased secretion. IL-6 transcription in response to ATP was not affected by Bapta or by the T channel blocker. Our results prove that ATP-stimulated IL-6 secretion is mediated at the post-transcriptional level by Ca²⁺ signals, including the mobilization of calcium stores, the activation of store-operated Ca²⁺ entry, and the subsequent activation of voltage-operated Ca²⁺ channels and calmodulin/calcineurin pathways.