Arginine kinase is closely associated with adaptation to environmental stresses such as high salinity and heavy metal ion levels in marine invertebrates. In this study, the effects of Cd²⁺ on the cuttlefish Sepia pharaonis’ arginine kinase (SPAK) were investigated. SPAK was isolated from the muscles of S. pharaonis and upon further purification, showed a single band on SDS-PAGE. Cd²⁺ effectively inactivated SPAK, and the double-reciprocal kinetics indicated that Cd²⁺ induced non-competitive inhibition of arginine and ATP. Spectrofluorometry results showed that Cd²⁺ induced tertiary structure changes in SPAK with the exposure of hydrophobic surfaces that directly induced SPAK aggregation. The addition of osmolytes, glycine, and proline successfully blocked SPAK aggregation and restored the conformation and activity of SPAK. Molecular dynamics simulations involving SPAK and Cd²⁺ showed that Cd²⁺ partly blocks the entrance of ATP to the active site, and this result is consistent with the experimental results showing Cd²⁺-induced inactivation of SPAK. These results demonstrate the effect of Cd²⁺ on SPAK enzymatic function and unfolding, including aggregation and the protective effects of osmolytes on SPAK folding. This study provides concrete evidence of the toxicity of Cd²⁺ in the context of the metabolic enzyme SPAK, and it illustrates the toxic effects of heavy metals and detoxification mechanisms in cuttlefish.