Hydrogel-based artificial muscle is of great interest for realizing diverse stimuli-induced actuations due to biocompatibility and tissue-like softness. Under physiological circumstances, calcium ions are an important cue for conveying in vivo signals such as skeletal muscle contraction. Here, we show that DNA artificial muscle (DAM) reversibly actuates in a calcium-dependent manner. A positively charged calcium initially takes part in the stabilization of DNA hydrogel fiber, and plays a role as a stimulator based on an ionic response to negatively charged phosphate residues of DNA for actuation. The actuations of DAM demonstrate a reversible responsiveness under calcium-controlled conditions. The twisted DAM rotated in the same as an initial twisting-direction and was affected by the length ratio of the actuating/nonactuating part, twist density, and calcium concentration. The tensile actuation of the coiled DAM was regulated by calcium concentration, twist density, and applied stress. Remarkably, the coiled DAM exhibited a prominent work performance (50.86 J/kg), which is higher than that of a natural muscle (40 J/kg). The current study presents reversible and calcium-dependent actuations of DAM similar to the contractile mechanism of natural muscle. Therefore, we suggest that the artificial muscles utilizing DNA offer the great potential for biomedical-based diverse applications.