We previously reported that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is inhibited by S-nitrosylation of Cys(6) in cells. Herein, we show that polysulfidation of CaMKII at Cys(6) limits its enzyme activity following reactive sulfur species (RSS) stimulus. In vitro incubation of CaMKII with the RSS donor, Na2S4, induced the inhibition of the enzyme via its polysulfidation. Treatment with dithiothreitol reversed the polysulfidation and the subsequent inhibition. The inhibition of CaMKII by Na2S4 is competitive with ATP but not with the peptide substrate Syntide-2. In transfected cells expressing CaMKII, the enzyme activity decreased upon treatment with Na2S4, whereas cells expressing mutant CaMKII (C6A) were resistant to this treatment. In addition, the endogenous CaMKII was inhibited by treatment with Na2S4 in RAW264.7 murine macrophage cells. These results suggest a novel regulation of CaMKII by RSS via its Cys(6) polysulfidation in cells. (C) 2018 Elsevier Inc. All rights reserved.