Sodium pyrithione (NaPT) is a biocide with fungistatic and antimicrobial properties, and is commonly employed as a zinc ionophore for research purposes. Despite biocidal effects on microorganisms, its biological and toxicological effects on mammalian cells remain poorly understood beyond ionophore activity. Thus, its cytotoxicity and underlying mechanisms were investigated in primary cultured vascular smooth muscle cells. Different from general notion, NaPT treatment alone, even in the absence of extracellular zinc, markedly increased cytosolic zinc levels. The cytotoxicity induced by prolonged NaPT treatment was prevented by the zinc chelator TPEN, indicating zinc a culprit for cytotoxicity. NaPT depleted cytosolic thiols, while supplementation with a thiol donor blocked zinc elevation, suggesting the zinc release from zinc-binding proteins. NaPT stimulated reactive oxygen species (ROS) production via protein kinase C-mediated activation of NADPH oxidase 1 (NOX1), independently of mitochondria. Both the NaPT-induced thiol depletion and zinc elevation were prevented by NOX inhibitors, NOX1 knockdown, or a ROS scavenger, demonstrating that NOX-derived ROS drive these effects. Consistently, NaPT-induced cytotoxicity was suppressed by NOX inhibitors or knockdown, ROS scavengers, and a thiol donor. Collectively, NaPT, regardless of its function as an ionophore, elevates cytosolic zinc by stimulating NOX-derived ROS formation and subsequent thiol depletion. Cytosolic free zinc thus acts as a crucial mediator of NaPT-induced cytotoxicity in mammalian cells.