The NPR1 or NPR1-like genes play a pivotal role in systemic acquired resistance in plants. Here, we isolated and identified a novel tobacco (Nicotiana glutinosa) NPR1-like gene (designated as NgNPR3). The full-length cDNA is 2049 bp in length with a 1767 bp open reading frame which encodes a 588 amino acids protein with an estimated molecular mass of 66 kDa and a calculated pI of 7.14. Homology analysis suggested that the NgNPR3 protein shares significant similarity to AtNPR3 of Arabidopsis. Transient expression assay of NgNPR3-GFP fusion gene in onion epidermal cells revealed that the NgNPR3 protein was localized to the cytoplasm and moved into the nucleus after redox change. RT-PCR results indicated that NgNPR3 was up-regulated after treatment with SA, INA, H2O2, and MeJA, which play important roles in various resistance responses in tobacco. Transcriptional level of NgNPR3 was also up-regulated after inoculation with Rhizoctonia solani, Phytophthora parasitica, Alternaria alternata, Pseudomonas solanacearum, and potato virus Y (PVY), respectively. When NgNPR3 was overexpressed in N. tabacum cv. Samsun plants, the transgenic plants showed enhanced resistance to the pathogens A. alternate, P. solanacearum and PVY. Furthermore, NgNPR3-mediated disease resistance is dosage-dependent. Our results suggest that NgNPR3 could be a putative NPR1-like gene, and might play an important role in resistance to a broad range of pathogens in tobacco.