Alkyl gallates showed elicitor activities on tobacco in both whole plants and cell suspensions. Methyl gallate (MG), ethyl gallate (EG), and propyl gallate (PG) infiltration into tobacco leaves induced hypersensitive reaction-like lesions and topical production of autofluorescent compounds revealed under UV light. When sprayed on tobacco plants at 5 mM, EG promoted upregulation of defense-related genes such as the antimicrobial PR1, beta-1,3-glucanase PR2, Chitinase PR3, and osmotin PR5 target genes. Tobacco BY-2 cells challenged with EG underwent cell death in 48 h, which was significantly reduced in the presence of the protease inhibitor aprotinin. The three alkyl gallates all caused alkalinization of the BY-2 extracellular medium, whereas gallic acid did not trigger any pH variation. Using EGTA or LaCl3, we showed that Ca2+ mobilization occurred in BY-2 cells elicited with EG. Overall, our findings are the first evidence of alkyl gallate elicitor properties with early perception events on the plasma membrane, potential hypersensitive reactions, and PR-related downstream defense responses in tobacco.