Understanding the molecular pathway(s) of antioxidant gene regulation is of crucial importance for developing antioxidant-inducing agents for the intervention of oxidative cardiac disorders. Accordingly, this study was undertaken to determine the role of Nrf2 signaling in the basal expression as well as the chemical inducibility of endogenous antioxidants and phase 2 enzymes in cardiac fibroblasts. The basal expression of a scope of key cellular antioxidants and phase 2 enzymes was significantly lower in cardiac fibroblasts derived from Nrf2-/- mice than those from wild type control. These include catalase, reduced glutathione (GSH), glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase-1 (NQO1). Incubation of Nrf2+/+ cardiac fibroblasts with 3H-1,2-dithiole-3-thione (D3T) led to a significant induction of superoxide dismutase (SOD), catalase, GSH, GR, glutathione peroxidase (GPx), GST, and NQO1. The inducibility of SOD, catalase, GSH, GR, GST, and NQO1, but not GPx by D3T was completely abolished in Nrf2-/- cells. The Nrf2-/- cardiac fibroblasts were much more sensitive to reactive oxygen and nitrogen species-mediated cytotoxicity. Upregulation of antioxidants and phase 2 enzymes by D3T in Nrf2+/+ cardiac fibroblasts resulted in a dramatically increased resistance to the above species-induced cytotoxicity. In contrast, D3T-treatment of the Nrf2-/- cells only provided a slight cytoprotection. Taken together, this study demonstrates for the first time that Nrf2 is critically involved in the regulation of the basal expression and chemical induction of a number of antioxidants and phase 2 enzymes in cardiac fibroblasts, and is an important factor in controlling cardiac cellular susceptibility to reactive oxygen and nitrogen species-induced cytotoxicity.