Oomycetes constitute an important agricultural pathogen for plants of the Solanaceae family. Black shank, caused by Phytophthora parasitica var. nicotianae, is one of the most important diseases affecting tobacco plantations in Cuba and worldwide, constituting an occasional cause of major economic losses. We have constructed a subtractive library with the aim of identifying tobacco genes involved in the resistance to this oomycete. The analysis by realtime PCR confirmed, for the first time, the patterns of differential expression between resistance and susceptibility reactions for the identified genes. This constitutes the first report demonstrating the pattern of expression of the glutathione-S-transferase (GST) gene in varieties and species independently of the degree of resistance or susceptibility to the pathogen. The gene was cloned in a plant transformation vector for gene silencing, and transgenic N. tabacum plants with silenced GST were obtained. They significantly displayed increased resistance to the disease, in highly susceptible plants, in comparison to control plants. This work constitutes the first report on the role of GST as a negative regulator of the defensive response to oomycetes, as well as the first instance where RNA interference technology has ever been used to obtain resistance to diseases produced by Oomycetes.