Nitric oxide (NO) is a ubiquitous signaling molecule involved in a wide range of physiological and pathophysiological processes in animals and plants. Although its significant influence on plant immunity is well known, information about the exact regulatory mechanisms and signaling pathways involved in the defense response to pathogens is still limited. We used genetic, biochemical, pharmacological approaches in combination with infection experiments to investigate the NO-triggered salicylic acid (SA)-dependent defense response in Arabidopsis thaliana. The NO donor S-nitrosoglutathione (GSNO) promoted the nuclear accumulation of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) protein accompanied by an elevated SA concentration and the activation of pathogenesis-related (PR) genes, leading to induced resistance of A. thaliana against Pseudomonas infection. Moreover, NO induced a rapid change in the glutathione status, resulting in increased concentrations of glutathione, which is required for SA accumulation and activation of the NPR1-dependent defense response. Our data imply crosstalk between NO and glutathione, which is integral to the NPR1-dependent defense signaling pathway, and further demonstrate that glutathione is not only an important cellular redox buffer but also a signaling molecule in the plant defense response.