Expression of the ZmNBS42 in Arabidopsis plants conferred resistance to bacterial pathogens, providing potential resistance enhancement of maize in further genetic breeding. Nucleotide-binding site (NBS) domain proteins play critical roles in disease resistance. In this study, we isolate a novel NBS gene ZmNBS42 from maize and systematically investigate its function on disease resistance. We find that the expression levels of ZmNBS42 in maize leaf were strikingly increased in response to Bipolaris maydis inoculation and SA treatment. The spatial expression pattern analysis reveals that, during development, ZmNBS42 is ubiquitously highly expressed in maize root, leaf, stem, internode and seed, but lowly expressed in pericarp and embryo. To better understand the roles of ZmNBS42, we overexpressed ZmNBS42 in heterologous systems. Transient overexpression of ZmNBS42 in the leaves of Nicotiana benthamiana induces a hypersensitive response. ZmNBS42 overexpression (ZmNBS42-OE) Arabidopsis plants produced more SA content than Col-0 plants, and increased the expression levels of some defense-responsive genes compared to Col-0 plants. Moreover, the ZmNBS42-OE Arabidopsis plants displayed enhanced resistance against Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000). These results together suggest that ZmNBS42 can serve as an important regulator in disease resistance, thus better understanding of ZmNBS42 would benefit the resistance enhancement in maize breeding programs.