Thiol antioxidants, including N-acetyl-l-cysteine (NAC), are widely used as modulators of the intracellular redox state. We investigated the hypothesis that NAC-induced reactive oxygen species (ROS) signaling perturbs cellular proliferation by regulating the cell cycle regulatory protein cyclin D1 and the ROS scavenging enzyme Mn–superoxide dismutase (MnSOD). When cultured in media containing NAC, mouse fibroblasts showed G₁ arrest with decreased cyclin D1 protein levels. The absence of a NAC-induced G₁ arrest in fibroblasts overexpressing cyclin D1 (or a nondegradable mutant of cyclin D1-T286A) indicates that cyclin D1 regulates this G₁ arrest. A delayed response to NAC exposure was an increase in both MnSOD protein and activity. NAC-induced G₁ arrest is exacerbated in MnSOD heterozygous fibroblasts. Results from electron spin resonance spectroscopy and flow cytometry measurements of dihydroethidine fluorescence showed an approximately 2-fold to 3-fold increase in the steady-state levels of superoxide (O₂^•−) in NAC-treated cells compared with control. Scavenging of O₂^•− with Tiron reversed the NAC-induced G₁ arrest. These results show that an O₂^•− signaling pathway regulates NAC-induced G₁ arrest by decreasing cyclin D1 protein levels and increasing MnSOD activity. [Cancer Res 2007;67(13):6392–9]