Background <p>Sonic Hedgehog (SHH) signaling pathway controls cell proliferation, differentiation, and organ formation. The changes in Ca²⁺ and their regulatory role in the context of activated SHH signaling have not been fully examined.</p> Results <p>In this study, we induced activation of the SHH signaling pathway using activators, including Full-length Shh protein (Shh) and N-terminal Shh (ShhN) ligands or a Smoothened agonist (SAG), while concurrently manipulating calcium level by either increasing extracellular Ca²⁺ concentration or employing BAPTA to chelate intracellular Ca²⁺. We found that the activation of SHH target genes by the three activators was not affected by low intracellular Ca²⁺ but inhibited by excess Ca²⁺. Although both Shh and SAG activate Ca²⁺ influx via the store-operated calcium entry (SOCE), their effects differ in the presence of BAPTA: Shh directly increases intracellular Ca²⁺ concentration, whereas the effect of SAG is enhanced by high extracellular Ca²⁺.</p> Conclusions <p>Excessive extracellular Ca²⁺ impairs SHH signal transduction, whereas intracellular Ca²⁺ levels have a negligible effect. At normal physiological extracellular Ca²⁺ concentrations, the depletion of intracellular Ca²⁺ does not affect SHH signal transduction.</p>