Chloroplast ATP synthase (cpATPase) is responsible for ATP production during photosynthesis. Our previous studies showed the cpATPase CF1 alpha subunit (AtpA) is a key protein involved in Clonostachys rosea (C. rosea)-induced resistance to the fungus Botrytis cinerea (B. cinerea) in tomato. Here, we show the expression level of tomato’s cpATPase CF1 alpha subunit gene (atpA) was up-regulated by B. cinerea and C. rosea. The tomato atpA gene was then isolated, and transgenic tobacco lines were obtained. Compared with untransformed plants, the atpA-overexpressing tobacco showed an increased resistance to B. cinerea, characterized by reduced disease incidence, defense-associated hypersensitive response (HR)-like reactions, balanced reactive oxygen species, alleviated damage to chloroplast ultra-structure of leaf cell, elevated levels of ATP content and cpATPase activity, enhanced expression of carbon metabolism-, photosynthesis-, and defense-related genes. Incremental Ca2+ efflux and steady H+ efflux were observed in transgenic tobacco after their inoculation with B. cinerea. Additionally, overexpression of atpA gene conferred enhanced tolerance to salinity and resistance to the fungus Cladosporium fulvum. Thus, the α subunit of cpATPase is a key regulator that links signaling to cellular redox homeostasis, ATP biosynthesis, and gene expression of resistance traits to modulate immunity to pathogen infection, in the process providing broad-spectrum resistance in plants.