A linkage between sulfur and iron metabolism has been suggested since sulfide has the ability to release iron from ferritin in the presence of iron acceptors in vitro. Nevertheless, this linkage is still lacking evidence in vivo as well as in cellular models. In this study we have treated human RD4 skeletal muscle cells with sodium sulfide and measured the level of the labile iron pool (LIP) as well as the intracellular sulfide concentration. We have also detected the amounts of L-ferritin protein as well as the iron regulatory protein 2 (IRP2). The sulfide treatment resulted in a 100% increase in the amount of LIP after 1 and 2 h. We also found that the raise of the LIP levels was coupled to an elevation of the amounts of intracellular sulfide that increased by 60%. The bioavailability of the released iron was confirmed by a 100% increase in L-ferritin protein as well as a 60% decrease of the IRP2 protein levels. These results suggest that there is a linkage between sulfur metabolism and intracellular iron regulation in mammalian cells.