The NPR1 (nonexpressor of pathogenesis-related genes 1) gene is an activator of the systemic acquisition of resistance (SAR) in plants and is one of the central factors in their response to pathogenic bacterial infestation, playing an important role in plant disease resistance. Potato (Solanum tuberosum) is a crucial non-grain crop that has been extensively studied. However, the identification and analysis of theNPR1-like gene within potato have not been understood well. In this study, a total of six NPR1-like proteins were identified in potato, and phylogenetic analysis showed that the six NPR1-like proteins inSolanum tuberosumcould be divided into three major groups with NPR1-related proteins fromArabidopsis thalianaand other plants. Analysis of the exon–intron patterns and protein domains of the sixNPR1-like genes from potato showed that the exon–intron patterns and protein domains of the NPR1-like genes belonging to the sameArabidopsis thalianasubfamily were similar. By performing quantitative real-time PCR (qRT-PCR) analysis, we found that six NPR1-like proteins have different expression patterns in different potato tissues. In addition, the expression of threeStNPR1genes was significantly downregulated after being infected byRalstonia solanacearum(RS), while the difference in the expression ofStNPR2/3was insignificant. We also established potatoStNPR1overexpression lines that showed a significantly increased resistance toR. solanacearumand elevated activities of chitinase, β-1,3-glucanase, and phenylalanine deaminase. Increased peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities, as well as decreased hydrogen peroxide, regulated the dynamic balance of reactive oxygen species (ROS) in theStNPR1overexpression lines. The transgenic plants activated the expression of the genes associated with the Salicylic acid (SA) defense response but suppressed the expression of the genes associated with Jasmonic acid (JA) signaling. This resulted in resistance toRalstonia solanacearum.