Previously, we reported that mitochondria-associated hexokinases are active in controlling programmed cell death in plants (Plant Cell 18, 2341-2355). Here, we investigated their role under abiotic- and biotic-stress conditions. Expression of NbHxk1, a Nicotiana benthamiana hexokinase gene, was stimulated by treatment with salicylic acid or methyl viologen (MV), and was also up-regulated by pathogen infection. In response to MV-induced oxidative stress, NbHxk1-silenced plants exhibited increased susceptibility, while the HXK1- and HXK2-overexpressing Arabidopsis plants had enhanced tolerance. Moreover, those overexpressing plants showed greater resistance to the necrotrophic fungal pathogen Alternaria brassicicola. HXK-overexpression also mildly protected plants against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000, a response that was accompanied by increased H2O2 production and elevated PR1 gene expression. These results demonstrate that higher levels of hexokinase confer improved resistance to MV-induced oxidative stress and pathogen infection.