Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is effective against a broad range of pathogens. SAR development in dicotyledonous plant such as tobacco and Arabidopsis is mediated by salicylic acid (SA). The signaling pathway upstream of SA accumulation is partially clarified inArabidopsis but little is known in other plant species. We investigated the SAR signaling pathway in tobacco using probenazole (PBZ) and its active metabolite, 1,2-benzisothiazole-1,1-dioxide (BIT), which induce SAR inArabidopsis through SA accumulation. Wild-type tobacco treated with PBZ or BIT exhibited increased expression of several pathogenesis related (PR) genes, increased levels of free and total SA, and enhanced resistance to the viral pathogen tobacco mosaic virus (TMV), the bacterial pathogen Pseudomonas syringae pv. tabaci (Pst), and the fungal pathogen Oidium sp. The role of SA in activating resistance following PBZ or BIT treatment was analysed using NahG transgenic plants. PBZ or BIT treatment did not induce disease resistance and PR-1 expression in NahG transgenic plants, which indicated that these compounds induce SAR by triggering signaling at a point upstream of SA accumulation. These results suggested that similar signaling pathways upstream of SA function in dicotyledonous plants species.