Iron (Fe) plays a critical role in proliferation, and Fe deficiency results in G₁/S arrest and apoptosis. However, the precise role of Fe in cell-cycle control remains unclear. We observed that Fe depletion increased the mRNA of the universal cyclin-dependent kinase inhibitor, p21^CIP1/WAF1, while its protein level was not elevated. This observation is unique to the G₁/S arrest seen after Fe deprivation, as increased p21^CIP1/WAF1 mRNA and protein are usually found when arrest is induced by other stimuli. In this study, we examined the posttranscriptional regulation of p21^CIP1/WAF1 after Fe depletion and demonstrated that its down-regulation was due to 2 mechanisms: (1) inhibited translocation of p21^CIP1/WAF1 mRNA from the nucleus to cytosolic translational machinery; and (2) induction of ubiquitin-independent proteasomal degradation. Iron chelation significantly (P < .01) decreased p21^CIP1/WAF1 protein half-life from 61 (± 4 minutes; n = 3) to 28 (± 9 minutes, n = 3). Proteasomal inhibitors rescued the chelator-mediated decrease in p21^CIP1/WAF1 protein, while lysosomotropic agents were not effective. In Fe-replete cells, p21^CIP1/WAF1 was degraded in an ubiquitin-dependent manner, while after Fe depletion, ubiquitin-independent proteasomal degradation occurred. These results are important for considering the mechanism of Fe depletion–mediated cell-cycle arrest and apoptosis and the efficacy of chelators as antitumor agents.