Plant non-symbiotic hemoglobins (nsHbs) play important roles in a variety of cellular processes. Previous evidence from this laboratory indicates that the expression of a class 1 nsHb gene (GhHb1) from cotton is induced in cotton roots challenged with the Verticillium wilt fungus. The present study examined further the expression patterns of the GhHb1 gene in cotton plants and characterized its in vivo function through ectopic overexpression of the gene in Arabidopsis thaliana. Expression of GhHb1 in cotton plants was induced by exogenously applied salicylic acid, methyl jasmonic acid, ethylene, hydrogen peroxide (H2O2) and nitric oxide (NO). Ectopic overproduction of GhHb1 in Arabidopsis led to constitutive expression of the defense genes PR-1 and PDF1.2, and conferred enhanced disease resistance to Pseudomonas syringae and tolerance to V. dahliae. GhHb1-transgenic Arabidopsis seedlings were more tolerant to exogenous NO and contained lower levels of cellular NO than the wild-type control. Moreover, transgenic plants with relatively high levels of expression of the GhHb1 gene developed spontaneous hypersensitive lesions on the leaves in the absence of pathogen inoculation. Our results indicate that GhHb1 proteins play a role in the defense responses against pathogen invasions, possibly by modulating the NO level and the ratio of H2O2/NO in the defense process.